CN106540258A

CN106540258A - Application of the Hippo paths in S100A7, the S100A8 and S100A9 expression of regulation and control cancerous cell

Info

Publication number: CN106540258A
Application number: CN201510608676.3A
Authority: CN
Inventors: 肖雪媛; 孔菲; 李允广; 王睿; 何大澄; 王俊豪; 胡恩泽
Original assignee: Beijing Normal University
Current assignee: Beijing Normal University
Priority date: 2015-09-22
Filing date: 2015-09-22
Publication date: 2017-03-29
Anticipated expiration: 2035-09-22
Also published as: CN106540258B

Abstract

The invention discloses application of the Hippo paths in S100A7, the S100A8 and S100A9 expression of regulation and control cancerous cell.The invention provides application of the material of YAP protein actives in following (a) or (b) can be suppressed：A () promotes S100A7 albumen and/or S100A8 albumen and/or S100A9 protein expressions in cancerous cell；B () is prepared for promoting the product of S100A7 albumen and/or S100A8 albumen and/or S100A9 protein expressions in cancerous cell.It is demonstrated experimentally that YAP protein actives in anticancer, can promote the expression of S100A7, S100A8 and S100A9 albumen in cancerous cell.The present invention discloses the rule of S100A7, S100A8 and S100A9 abduction delivering in various cancerous cell first, and the medicine of the tumor related to S100A7, S100A8 and S100A9 to design treatment provides important foundation, while the occurrence and development to illustrating tumor provide important theoretical basiss.

Description

Application of the Hippo paths in S100A7, the S100A8 and S100A9 expression of regulation and control cancerous cell

Technical field

The invention belongs to biological technical field, is related to Hippo paths in regulation and control cancerous cell S100A7, S100A8 and S100A9 Application in expression, the more particularly to activity of YAP albumen are in S100A7, the S100A8 and S100A9 expression of regulation and control cancerous cell Application.

Background technology

Hippo paths are most found in fruit bat, and under physiological statuss, Hippo paths are mainly the propagation of regulating cell and wither Die and limit the size that histoorgan grows, and participate in the self renewal of stem cell.Find in the recent period, Hippo signal pathways are one The new tumorigenic signal path of suppression is planted, and YAP/TAZ is the core protein of Hippo signal pathways.In mammal In, activity of the Hippo paths by 4 main kinase regulatory YAP.Cause Mst1/2 after Hippo signal paths are activated Activation, Mst1/2 kinases make Lat1/2 (Large tumor suppressor with scaffolding protein Sav1 composition complex；LATS) swash Enzyme phosphorylation is simultaneously activated, and the Lat1/2 kinases of phosphorylation constitutes complex with another scaffolding protein Mob1 again.Lat1/2 is direct Make after S127 is phosphorylated on YAP albumen, to produce the binding motif of a 14-3-3, so as to can be with the 14-3-3 albumen in endochylema With reference to being trapped in endochylema, cause YAP to enter to examine and make cuts less, or be degraded by proteasome pathway after being further phosphorylated. As YAP does not have DNA binding activity.Therefore, the YAP into the non-phosphorylating state of core is combined with other transcription factor Competence exertion its biological action.At present it is generally accepted that TEAD and p73.YAP and transcription factor TEAD in core (TEA-domain；TEAD) combine, promote or suppress the expression of TEAD target genes, such as CTGF and Cyr61, so as to adjust Section various biological function, such as cell growth, cells contacting suppress, control the size of histoorgan and the self renewal of stem cell Deng.TAZ is the homologous protein of YAP, has same or similar biological action with YAP, such as also can be by LATS1/2 phosphoric acid Change, the TAZ for entering core also can be combined the expression for promoting CTGF and CYR61 with TEAD.In addition YAP also can be combined with p73 Regulation and control and the expression of gene participating in apoptosis, such as Bax and DR5 etc..In addition also have been reported that, find in kinds of tumors tissue The expression of YAP in core is increased, and this suggests that the activity of YAP is closely related with the occurrence and development of tumor.The activity of YAP is removed Adjusted outer by Hippo paths, also reported other kinases also scalable YAP phosphorylation activity in recent years successively.As in endochylema Further by CK1 δ/ε phosphorylations, and which can be made to degrade by proteasome after YAP S127 phosphorylations.Although this field is just Develop rapidly, but relevant YAP regulates and controls the research of the albumen related to cancer, such as S100A7, S100A8 and S100A9 albumen Research have no any report.

S100A7 also known as silver bits are plain (psoriasin), isolated in the epidermal keratinocyte of hypertrophy in initial psoriasiss.Its base Because, on the EDC regions of human chromosome 1q21, being made up of with two introns three exons.Research thinks, S100A7 Express in Skin Cell and secrete, antibacterial peptide (antimicrobial peptide) effect for killing antibacterial can be played, skin is helped Cell resists the invasion of the pathogenic bacterias such as escherichia coli, and as immune chemotactic factor, attracts CD4⁺Lymphocyte and neutrophilic granulocyte etc. are joined With inflammatory reaction.S100A7 great expressions in psoriasiss, therefore S100A7 also known as psoriasin.Additionally, S100A7 is in bag Overexpression in various dermatosis such as atoipc dermatitis, cutaneous T cell lymphoma, lichen sclerosus et atrophicuss is included, shows which in skin May play a role in differentiation.It has now been found that, S100A7 can be induced by a wide variety of factors expression.In normal epithelial tissues S100A7 expression is relatively low, but can be tested (experimental barrier by skin injury and experimental barrier breakdown by various Disruption) induced.And in the keratinocyte (keratinocytes) of In vitro culture, E. coli lysate, LPS The proinflammatory factor such as (e. coli lipopolysaccharide), interleukin (Interleukin), ultra-vioket radiation, 2mM calcium ions, dimension The rush differentiation such as formic acid, confluence condition of culture, suspension culture, serum are hungered and thirst, TPA factor all can induce S100A7 expression. In galactophore epithelial cell system MCF10A, S100A7 decapacitation is promoted outside the induction of differentiation factor by confluence and suspension culture etc., Can also be induced by active oxygen (reactive oxygen species), and be suppressed by antioxidant such as NAC, show S100A7 May be relevant with cell oxidative burst.In cancerous cell, the research of S100A7 abduction deliverings is still less, but there are some researches show, In breast cancer cell line MB-231, can be by DNA damage induced by chemotherapeutic agents, and in human mouth squamous cell carcinoma system, S100A7 Can be induced by confluence and suspension culture.Research discovery, S100A7 abnormal expressions in kinds cancer, and may be in cancer Disease plays a significant role in there is development.In breast carcinoma, expression is raised S100A7 in cancer in the original location, and is presented with patient's prognosis Dependency.The research of Emberley ED et al. finds that, in breast cancer cell line, S100A7 can be with Jab1 (c-jun 1) direct interaction, promotion Jab1 enter core to activation-domain binding protein, and activation AP-1 activity is final to be reduced The expression of p27 (a kind of cell cycle arrest factor), so as to promote cell to breed.In breast cancer cell line, Liu H et al. Also, it was found that S100A7 can promote NF-KB to enter core, promote cell growth.But in Dental clinic, S100A7 The expression in early stage hypertrophy and differentiated cancer is raised, and in low differentiation and metastatic carcinoma expresses low.Research finds Dental clinic In system, overexpression S100A7 can promote cell differentiation by suppressing β-catenin signal paths, so as to anticancer is bred. But after studying also, it was found that β-catenin signal paths are activated, S100A7 can be suppressed to express, mitigate cell differentiation, be bred Accelerate.In transitional cell bladder carcinoma, it is found that S100A7 protein positive rates significantly rise, show S100A7 with as bladder The potentiality of cancer molecular diagnosis mark.In ovarian cancer, research it is again seen that S100A7 expression versus normal tissues be increased significantly, And can be used as the molecular marker of cancer diagnosis.

S100A8/S100A9 genes are equally positioned on the EDC regions of chromosome 1q21 with S100A7, and gene is all by three Exon and two introns are constituted.S100A8/A9 protein structures are similar to, also by two EF- palmistrys near N-terminal and C-terminal Domain and central hinge area are constituted, and molecular weight is respectively 11KDa/13KDa.What S100A8/S100A9 was often relied on calcium ion Mode is formed in the form of heterodimer albumen composition.Both albumen are thin in vain in medullary cell, mononuclear cell, graininess It is in constitutive expression in born of the same parents, osteoclast.In addition, S100A8/A9 is in coexpression in many organizing.S100A8/S100A9 Several functions can be played in normal cell.Research finds that, after knocking out S100A8, mice embryonic can not be survived, this explanation S100A8 Irreplaceable important function is played in vivo.In macrophage, dendritic cell and microvascular endothelial cells, S100A8 can By inflammatory stimulus abduction delivering, and in neutrophilic granulocyte Cytoplasm, S100A8 protein contents account for as many as total protein concentration 20%, show Which may play a role in inflammatory reaction and immunity of organism effect.In addition study in also finding mouse epithelial cells, oxidative stress can Stimulate S100A8 expression.S100A9 equally plays a role in inflammatory reaction with immune system.Research shows that S100A9 exists Neutrophilic granulocyte can be attracted as chemotactic factor in human body and stimulate its synthesis integrin, so as to promote neutrophilic granulocyte to tissue The adhesion of middle fibronectin.There is research it is also shown that S100A9 can be by activating NF- κ B signal paths in macrophage The expression of middle induction TNF-α and interleukin, and TLR-4 receptor activations neutrophilic granulocyte can be passed through so as to adjust immune system. Another critical function of S100A9 is that it can remove activity in vivo oxygen as street cleaner, protects cells from oxygen injury.Except list Solely outside function, S100A8/A9 can play wider function after heteromeric complexes are formed.Such as S100A8/A9 again Compound can suppress the activity of Casein kinase 1/2, adjust marrow hemopoietic stem cells differentiation, help intracellular unsaturated fatty acid Transport, and P67 and RAC-2 interact and the several functions such as NAPDH oxidase activated in macrophage.S100A8 and S100A9 also plays an important role in cancer occurs development.In gastric cancer, S100A8 and S100A9 up-regulateds are found, Intra-tumor Interstitial cell and inflammatory cell great expression and secretion S100A8/A9 in gastric cancer, and S100A8/A9 by increasing P38 The phosphorylation of MAPK, activates NF-KB signal paths, and then the expression of increase MMP2/MMP12, so as to increase tumor Invasive ability.Also there are some researches prove, in gastric cancer, S100A8/A9 is expressed and secreted to immunosuppressant cell (MDSCs), And then suppress attack of the immune system to cancerous cell.In pulmonary carcinoma, S100A8/A9 is in adenocarcinoma and terminal cancer in notable expression Adjust, and it is relevant with clinical prognosis.Hiratsuta et al. has found that primary tumo(u)r is by inducing the secretion of metastasis normal cell in pulmonary carcinoma S100A8 and S100A9, attracts MaC-1 positive macrophages, and S100A8/A9 equally can pass through in tumor cell Activation MAPK and P38 signal paths, increase tumor invasion and metabasis ability^[24].In breast carcinoma, research then finds S100A8/A9 May play a role in drug resistant tumor cells, SwarmaliAxharyya et al. has found, although chemotherapeutics can be killed and be pressed down Growth of cancer cells processed, but while chemotherapy also makes endotheliocyte produce TNF-α.TNF-α is enhanced swollen by NF- κ B signal paths The expression of CXCL1/2 in oncocyte, so as to recruit CD11b (+) Gr1 (+) myelocyte that can express CXCR2, myelocyte Secretion S100A8/A9 promotes proliferative activity o f tumor and transitivity to increase, and counteracts chemotherapeutics effect.In hepatocarcinoma, S100A8 and S100A9 has obvious up-regulated expression in people and rat liver cancer tissue, and thinks which can pass through NF-KB signals Path changes in cancerous cell activity keto concentration so as to increasing cancerous cell survival ability.In colon cancer, research find S1100A8 and A9 can activate Smad4 signal paths by phosphorylation smad2/3, promote cancer cell migration and propagation.Additionally, in ovary In the kinds cancers such as cancer, carcinoma of gallbladder, carcinoma of prostate, cancer of pancreas, leukemia, there are document report S100A8 and S100A9 expression It is not normal and cancer occur development in work.In sum, the S100A8 and S100A9 high expression in kinds of tumor cells, And the growth and migration of mediate tumor cell.

The content of the invention

It is an object of the invention to provide the new application of YAP albumen and its activity inhibitor and accelerative activator.

The invention provides application of the material of YAP protein actives in following (a) or (b) can be suppressed：

A () promotes S100A7 albumen and/or S100A8 albumen and/or S100A9 protein expressions in cancerous cell；

B () is prepared for promoting the product of S100A7 albumen and/or S100A8 albumen and/or S100A9 protein expressions in cancerous cell Product.

Active component is the material that can suppress YAP protein actives, can be used in promote cancerous cell in S100A7 albumen and/or The product of S100A8 albumen and/or S100A9 protein expressions falls within protection scope of the present invention.

Present invention also offers any one application in following (c) or (d) in following (1)-material shown in (5)：

(1) YAP albumen；

(2) S127A-YAP albumen；The S127A-YAP albumen is that the 127th serine of the YAP albumen is dashed forward It is changed into the albumen of gained after alanine；

(3) encoding gene of the YAP albumen, or expression cassette, the recombinant vector of the encoding gene containing the YAP albumen Or transgenic cell；

(4) encoding gene of the S127A-YAP albumen, or the table of the encoding gene containing the S127A-YAP albumen Up to box, recombinant vector or transgenic cell；

(5) material of YAP protein actives can be strengthened；

S100A7 albumen and/or S100A8 albumen and/or S100A9 protein expressions in (c) anticancer；

D () is prepared for S100A7 albumen in anticancer and/or S100A8 albumen and/or S100A9 protein expressions Product.

Active component is any one in following (1)-material shown in (5), and can be used in S100A7 eggs in anticancer The product of white and/or S100A8 albumen and/or S100A9 protein expressions falls within protection scope of the present invention：

(1) YAP albumen；

(5) material of YAP protein actives can be strengthened.

In the present invention, the material that can suppress YAP protein actives can be any one in following (a1) and (a2)：

(a1) it is capable of the nucleic acid molecules of the encoding gene of silence YAP albumen；

(a2) material of YAP protein phosphorylations can be promoted.

In the present invention, the material that can strengthen YAP protein actives can be the material that can suppress YAP protein phosphorylations.

Further, the nucleic acid molecules of the encoding gene for being capable of silence YAP albumen are specially sequence in sequence table 3 and sequence Two single-chain nucleic acids shown in row 4 are annealed the double-strandednucleic acid to be formed；

The material that YAP protein phosphorylations can be promoted can in following any one：LATS1 albumen, encodes the LATS1 The encoding gene of albumen, the expression cassette of the encoding gene containing the LATS1 albumen, recombinant vector or transgenic cell, LATS1 protein active accelerator；

The material that YAP protein phosphorylations can be suppressed can be LATS1 protein active inhibitor；Specifically, the Lats1 Protein active inhibitor is the nucleic acid molecules for being capable of the encoding gene of Lats1 albumen described in silence；More specific, " the energy The nucleic acid molecules of the encoding gene of Lats1 albumen described in enough silences " are by two lists shown in sequence in sequence table 7 and sequence 8 The double-strandednucleic acid that chain Nucleic acids anneal is formed.

Following methods I or method II fall within protection scope of the present invention：

Method I：The method for promoting S100A7 albumen and/or S100A8 albumen and/or S100A9 protein expressions in cancerous cell, Comprise the steps：Suppress YAP protein actives in the cancerous cell, so as to realize promoting S100A7 albumen in the cancerous cell And/or the expression of S100A8 albumen and/or S100A9 albumen；

In methods described I, suppress YAP protein actives described in the cancerous cell be by following (b1)-(b3) at least It is a kind of to realize：

(b1) the previously described material that can suppress YAP protein actives is imported in the cancerous cell；

(b2) cancerous cell described in culture in the way of suspension culture；

(b3) cancerous cell described in culture in the way of over-confluence adhere-wall culture, and the culture density of the cancerous cell is 75000-150000 cell/cm²Culture area.

In (b2), the time of the suspension culture is more than 48 hours, such as 48-72 hours, 48 hours for another example.

Method II：The method of S100A7 albumen and/or S100A8 albumen and/or S100A9 protein expressions in anticancer, Comprise the steps (A) or (B) or (C), so as to realize suppressing S100A7 albumen and/or S100A8 in the cancerous cell The expression of albumen and/or S100A9 albumen：

(A) encoding gene of YAP albumen is imported in the cancerous cell, the expression of YAP albumen in the cancerous cell is improved Amount；

(B) encoding gene of S127A-YAP albumen is imported in the cancerous cell, makes the cancerous cell expression described S127A-YAP albumen；The S127A-YAP albumen be by the 127th mutant serine of the YAP albumen be alanine Albumen obtained by afterwards；

(C) YAP protein actives in the cancerous cell are strengthened；

In (C), it is by importing above in the cancerous cell to strengthen YAP protein actives described in the cancerous cell The described material that can strengthen YAP protein actives is realizing.

In the present invention, in the aminoacid sequence such as sequence table of the YAP albumen shown in sequence 1；The volume of the YAP albumen In the nucleotide sequence such as sequence table of code gene shown in sequence 2.Accordingly, the aminoacid sequence of the S127A-YAP albumen As the 127th serine by sequence in sequence table 1 is replaced with shown in gained sequence after alanine；The S127A-YAP eggs The oligonucleotide " TCC " of the 379-381 positions of sequence in sequence table 2 is such as replaced with by the nucleotide sequence of white encoding gene " GCC " is afterwards shown in gained sequence.

In the aminoacid sequence such as sequence table of the LATS1 albumen shown in sequence 5；The encoding gene of the LATS1 albumen In nucleotide sequence such as sequence table shown in sequence 6.

In the aminoacid sequence such as sequence table of the S100A7 albumen shown in sequence 9；The aminoacid sequence of the S100A8 albumen Row are as shown in sequence 10 in sequence table；In the aminoacid sequence such as sequence table of the S100A9 albumen shown in sequence 11.

In the present invention, the YAP albumen phosphatization acid is the 127th serine of protein shown in sequence 1 in sequence table Phosphorylation.

In the present invention, the cancerous cell can in following any one：Lung carcinoma cell, squamous carcinoma of the cervix cell, epidermis cancerous cell and Dental clinic.Further, the lung carcinoma cell concretely NCI-H292 cells or H226Br cells；The cervix uteri Squamous cell carcinoma concretely HCC94 cells；The epidermis cancerous cell concretely A431 cells；The Dental clinic tool Body can be FaDu cells.

Methods described I is preparing S100A7 albumen and/or S100A8 albumen and/or the application in S100A9 albumen falls within this The protection domain of invention.

In the present invention, the above all of product can be medicine.

It is demonstrated experimentally that YAP protein actives in anticancer, can promote S100A7, S100A8 and S100A9 in cancerous cell The expression of albumen.The present invention discloses the rule of S100A7, S100A8 and S100A9 abduction delivering in various cancerous cell first Rule, and the medicine of the tumor related to S100A7, S100A8 and S100A9 to design treatment provides important foundation, together When to illustrate tumor occurrence and development provide important theoretical basiss.

Description of the drawings

Fig. 1 is that SABC detects the expression of S100A7, S100A8 and S100A9 in various cancerous cell.Wherein, Br represents H226Br cells.It is dyed in figure tan for positive cell.

Fig. 2 can promote the expression of S100A7, S100A8 and S100A9 in cancerous cell for silence YAP albumen.Wherein, NC represents the compared with control cells transfected without si-YAP.

Fig. 3 is that the activity for improving YAP albumen can suppress the expression of S100A7, S100A8 and S100A9 in cancerous cell.Its In, NC represents the compared with control cells of suspension culture.Suspension represents suspension culture.H292 cells represent NCI-H292 Cell.

Fig. 4 is the shadow that cellular morphology is expressed in cancerous cell to the activity and its S100A7, S100A8 and S100A9 of YAP Ring.Wherein, A is q-PCR results；B is Western blot results, and NC represents normal density adhere-wall culture 48h, S48h Or S48 represents suspension culture 48h.H292 cells represent NCI-H292 cells.

Fig. 5 is activity and its expression of S100A7, S100A8 and S100A9 that cell density regulates and controls YAP.Wherein, A is Q-PCR results；B be Western blot results, L represent initial cell culture density be 7500 cells/cm²；H1 is represented just Beginning cell culture density is 100000 cells/cm²；H2 represents that initial cell culture density is 120000 cells/cm²；H3 is represented Initial cell culture density is 150000 cells/cm²；H represents that initial cell culture density is 75000 cells/cm²；con15d After representing that cell is covered with, then continuously cultivate 15 days, and the culture fluid for more renewing daily.H292 cells represent NCI-H292 Cell.

Fig. 6 is impacts of the LATS1 to S100A7, S100A8 and S100A, 9 expression in cancerous cell.A and B To S100A7, S100A8 and S100A9 expression after overexpression LATS1 respectively in HCC94 cells and A431 cells Impact.To S100A7, S100A8 and S100A9 after silence LATS1 in C and D respectively HCC94 cells and A431 cells The impact of expression.Wherein, Pcmv-nc represents the matched group for proceeding to pCMV-14 empty carriers；+ lats1 represents overexpression LATS1 Group；Sinc represents that siNC is compareed；SiLATS represents silence LATS1；Attached represents adhere-wall culture；Suspension is represented Suspension culture；Sparse represents low-density adhere-wall culture；Dense represents high density adhere-wall culture.

Fig. 7 be various cancerous cell to nude mice into tumor after tumor tissues ImmunohistochemistryResults Results.It is dyed in figure tan for the positive Cell.Wherein, Br represents H226Br cells.H292 cells represent NCI-H292 cells.

Specific embodiment

Experimental technique used in following embodiments if no special instructions, is conventional method.

In following embodiments, material used, reagent etc., if no special instructions, commercially obtain.

In following embodiments, all of quantitative experiment result is the result meansigma methodss of parallel laboratory test.All of experiment is repeated two It is secondary and more than twice.Experimental group and their corresponding matched groups carry out double tail T inspections using GraphPad softwares.Wherein, P Value is considered as significant difference less than 0.05.

NCI-H292 cells：Be recorded in " Tang Wei. poly L-arginine is to NCI-H292 cellular inflammation medium adjustment effects Research. Medical University Of Anhui, Master's thesis in 2014 " it is one literary, the public can be obtained from applicant, only limited the use of in repeating the present invention Experiment is used.

HCC94 cells：It is recorded in that " Yang Cheng, Rao Xiang, string .Twist genes are transfected to human cervical carcinoma cell biological behaviour shadow Loud observation. Chinese treatment and prevention of tumour magazine, 03 phase in 2013 " it is one literary, the public can be obtained from applicant, only limited the use of in repeating Present invention experiment is used.

A431 cells：Be recorded in " to open refined. nuclear hormone receptor is in skin main composition cell and epidermis scale cancer A431 cells Expression and its mediating effect+6 of some ligands. Third Military Medical University, thesis for the doctorate in 2013 " it is one literary, the public can be obtained from applicant , only limit the use of and use in repetition present invention experiment.

FaDu cells：Be recorded in " Lu Wuhao .HPV-16 infect the biological behaviour of laryngopharynx scale cancer and FaDu cells is affected and The regulation and control of miRNAs expression. Zhengzhou University, thesis for the doctorate in 2013 " it is one literary, the public can be obtained from applicant, only limit the use of in Repeat present invention experiment to use.

H226Br cells：It is recorded in " Chiu-Chin Hwang, Kang Fang, Limin Li, et al.Insulin-like growth factor-I is an autocrine regulator for the brain metastatic variant of a human non-small cell lung cell Line.Cancer Letter, 94 (1995) 157-163 " one is literary, and the public can be obtained from applicant, only limits the use of real in the present invention is repeated Test use.

A431, NCI-H292, HCC94 cell strain is using 1640 culture medium culturings containing 10% (volume fraction) FBS, FaDu MEM culture medium culturing of the cell strain containing 10% (volume fraction) FBS, H226Br cells are using containing 10% (volume fraction) The DMEM culture medium of FBS, condition of culture 5%CO₂, 37 DEG C of constant temperature.

The present invention relates to following each albumen：(1) YAP albumen, in aminoacid sequence such as sequence table shown in sequence 1, coding In gene such as sequence table shown in sequence 2.(2) S127A-YAP albumen, aminoacid sequence is such as by the of sequence in sequence table 1 127 serines are replaced with after alanine shown in gained sequence；The nucleotide sequence of encoding gene is as by sequence 2 in sequence table The oligonucleotide " TCC " of 379-381 positions replace with " GCC " afterwards gained sequence shown in.(3) LATS1 albumen, amino In acid sequence such as sequence table shown in sequence 5；In the nucleotide sequence such as sequence table of encoding gene shown in sequence 6.(4)S100A7 Albumen, in aminoacid sequence such as sequence table shown in sequence 9；(5) S100A8 albumen, sequence in aminoacid sequence such as sequence table Shown in 10；(6) S100A9 albumen, in aminoacid sequence such as sequence table shown in sequence 11.

The expression of embodiment 1, S100A7, S100A8 and S100A9 albumen in cancerous cell

First, the present inventor have detected various cancerous cell (H226Br cells, NCI-H292 using the method for SABC Cell, HCC94 cells, A431 cells and FaDu cells) (cell of every kind of adhere-wall culture is referred under regular culture conditions The strain cell density required when daily passing on is carried out) expression of S100A7, S100A8 and S100A9 albumen in cell.

Specifically operate as follows：

Cell climbing sheet to be tested is taken, PBS is washed once, 3.4% (3.4g/100ml) formaldehyde (PBS matches somebody with somebody) room temperature fixes 20min, The penetrating 10min of PBS-0.5% (volume fraction) Triton room temperatures, PBS are washed once afterwards with 3% (volume fraction) hydrogen peroxide-PBS Incubation at room temperature 15min fire extinguishing Endogenous peroxidases, PBS are washed and are once sealed with 3% (volume fraction) Ox blood serum-PBS room temperatures afterwards 20min is closed, subsequently incubation one resists overnight or 37 DEG C of 1h, PBS to wash 3 times (each 10min), and be incubated HRP labellings two resist, 15min room temperatures, PBS are washed 3 times (each 5min), and after DAB colour developing 3min, haematoxylin is redyed, and is washed, and graded ethanol takes off Water, the transparent rear mounting of dimethylbenzene, basis of microscopic observation staining conditions.

Wherein, for detecting that the one of S100A7 albumen resists for rabbit-anti people S100A7 (Psoriasin) antibody (Santa Cruz Biotechnology Products, catalog number are SC67047)；Two resist for fast-type enzyme mark goat-anti rabbit/Mus IgG polymer (stepping novel agent Products, catalog number is KIT-5010).For detecting that the one of S100A8 albumen resists for anti-hS100A8 (R＆B Products, catalog number are MAB4570)；Two resist for fast-type enzyme mark goat-anti rabbit/Mus IgG polymer (step Novel agent Products, catalog number are KIT-5010).For detecting that the one of S100A9 albumen resists for goat-anti people S100A9 (Calgranulin B) antibody (Santa Cruz Biotechnology Products, catalog number is SC-8114)；Two Resist for fast-type enzyme mark rabbit-anti sheep IgG polymer (stepping novel agent company, catalog number is KIT-5107).

As a result show, (H226Br is thin in the cancerous cell line of above-mentioned detection for S100A7, S100A8 and S100A9 egg positive cell Born of the same parents, NCI-H292 cells, HCC94 cells, A431 cells and FaDu cells) in have expression, but ratio is very low. S100A7, S100A8 in addition to 10% or so, in other cancerous cell of the positive cell ratio of S100A7 in HCC94 cells With the positive cell ratio of S100A8 and S100A9 in S100A9 and HCC94 cells<1% (Fig. 1).

The expression of YAP albumen in embodiment 2, anticancer can promote the expression of S100A7, S100A8 and S100A9

Various cancerous cell lines, including lung adenocarcinoma cell line NCI-H292, squamous carcinoma of the cervix cell are transfected using the si-YAP of specificity Strain HCC94, epidermis JEG-3 A431 and Dental clinic strain FaDu.Using RT-PCR and/or western blot sides Method confirms the silencing efficiency of YAP.Then using RT-PCR and Western blot detection silences YAP after, S100A7 in cancerous cell, The expression of S100A8 and S100A9.

First, si-YAP transfected cancer cells system

In 700,000 cancerous cell of 60mm culture dishs middle berth, 24h is cultivated.By transfection reagent (jetPRIME), Buffer and use In interference YAP genes (sequence 2, Genbank：NM_001130145.2 the siRNA for) expressing is mixed, and stands 10min, Uniformly it is added drop-wise in culture medium.Cell is received after 48h standby.

Wherein, for disturbing YAP genes (sequence 2, Genbank：NM_001130145.2 the siRNA for) expressing is by such as The double-strand of two single-stranded annealing shown in lower A and B.

A：5 '-GGUGAUACUAUCAACCAAATT-3 ' (sequence 3)；

B：5 '-UUUGGUUGAUAGUAUCACCTT-3 ' (sequence 4).

2nd, YAP silencing efficiencies detection

1st, RT-PCR detections YAP silencing efficiencies

(1) cell total rna extracts the preparation with cDNA

The cell obtained after taking step one transfection, pre-cooling PBS are cleaned three times, are added 1mL Trizol, are blown with liquid-transfering gun Suction makes cell crack completely, and Trizol lysates are placed in EP pipes, is incubated at room temperature 5min；200 μ L chloroforms are added, is vortexed Concussion is mixed, and is incubated at room temperature 3min, is stood 15min on ice；4 DEG C of centrifugation 20min of 12000rpm, suct clear to another EP Guan Zhong；500 μ L isopropanols are added, is slowly mixed to solution turned clear, is stored at room temperature 10min；4 DEG C of 12000rpm, Centrifugation 10min, sucks supernatant；Plus 1ml dehydrated alcohol, overturn and mix, 4 DEG C, 12000rpm centrifugation 10min draw supernatant； Plus DEPC water matches somebody with somebody 75% ethanol, overturn and mix, 4 DEG C, 12000rpm centrifugation 10min suck supernatant；Dry up in super-clean bench Become RNA powder to EP bottom of the tube precipitation is transparent, can be frozen in -20 DEG C.

Appropriate DEPC water dissolutioies are added in RNA dry powder, its concentration are determined using ultraviolet spectrophotometer；Use DEPC water RNA is diluted to into 1 μ g/ μ L, following reaction system is prepared：2 μ L of RNA solution；Oligo(dT)₁₅0.5μL；DEPC water 2.5μL。

72 DEG C of liquid mixed above, 5min, the time terminates to take out immediately place on ice 5min, adds following reagent：5×M-MLV RT buffer 4μL；dNTPs 8μL；2 μ L of DEPC water；1 μ L of M-MLV reverse transcription.

42 DEG C of liquid mixed above, 60min；72 DEG C, 15min；CDNA synthesizes and completes, and is stored in 4 DEG C or frozen in -20 DEG C refrigerator is stand-by.

(2) RT-PCR detections gene expression

With GAPDH as internal reference, 4, each sample is parallel, and in negative control, cDNA templates are replaced with tri-distilled water.

Wherein, for expanding YAP gene (Genbank：NM_001130145.2 primer sequence) is as follows：

YAP-F：5’-CCTCTATTTTGCTCTTCCTTGTCC-3’；

YAP-R：5’-CCATCATCCAAACAGGCTCAC-3’.

Primer sequence for expanding internal reference GAPDH is as follows：

GAPDH-F：5’-GAGTCAACGGATTTGGTCGT-3’；

GAPDH-R：5’-GACAAGCTTCCCGTTCTCAG-3’.

Reaction system：10 μ L SYBR, 2 μ L primers, 7 μ L sterilized water, 1 μ L cDNA.

Response procedures：The first step：95℃10min.Second step：94 DEG C of 15s, 60 DEG C of 1min, 40 circulations.

Experiment is arranged without the corresponding cell of si-YAP transfections simultaneously as control.

(3) result

As a result it is as shown in Figure 2, it is seen that in each cancerous cell obtained after step one transfection, YAP is on rna level and without si-YAP The control of transfection is compared expression and is substantially reduced.

2nd, western blot detections YAP silencing efficiencies

(1) extraction of total protein of cell

The each cell obtained after taking step one transfection, pancreatin digestion (if suspension cell is then directly centrifuged), 1000rpm centrifugations 5min sedimentation cells；The PBS of pre-cooling is washed three times, and cell precipitation is collected to EP pipes；Add what 3-5 times of precipitation volume was measured RIPA lysates, piping and druming are mixed to acellular agglomerate, 4 DEG C of suspension 1h on suspension instrument；4 DEG C, 12000rpm centrifugation 40min, The supernatant liquid drawn in the middle part of centrifugation product is protein extract, subsequently uses Bradford method detection sample protein concentrations；To albumen 6 × protein loading buffer of 1/5 volume are added to mix in extracting solution, boiling water bath 5min makes protein disulfide bond open and become Property, after subpackage, -20 DEG C save backup.

(2) immunoblotting (Western blot) experiment

First, the Tricine-SDS-Page glue of configuration 12%, formula are following (by taking one piece of glue as an example)：AB-32.67ml；3 ×Gel buffer 3.33ml；Glycerol 1g；Pure water 4ml.50 μ l of 10%AP, 5 μ l of TEMED is added slightly to mix after stirring and evenly mixing Even rear placing separation gel, hydraulic pressure glue, after gelling to be separated, according still further to following recipe configuration concentration glue (as a example by one piece of glue)：AB-3 0.25ml；3×Gel buffer 0.75ml；Pure water 2ml.20 μ l of 10%AP, 2 μ l of TEMED is added slightly to mix after stirring and evenly mixing Even rear placing sample glue.

Negative electrode is separately added into up and down in glue groove after the completion of glue configuration, and anode buffer liquid is taken on equal protein sample and albumen Marker Sample, first 30V constant pressures electrophoresis treat that sample is fully entered in glue, then 80V constant pressures carry out sample gel electrophoresis, after sample enters separation gel Voltage is enlarged to 100 volts of electrophoresis 90min；Separation gel is taken out and cut, albumen is gone to into PVDF using the Bio-Rad wet instrument that turns On film (methanol is previously active), 350mA constant current transferring films 2h；Film room temperature is sealed with the 5% defatted milk powder solution that TBS is configured Close 1h；The one of destination protein is added to resist, 1h or 4 DEG C of overnight incubation of 37 DEG C of incubations in wet box；TBS washes film 3-5 time, and every time 5 min；Add two anti-, 37 DEG C of incubation 1h of the one anti-kind of correspondence of HRP labellings；TBS washes TBS and washes film 3-5 time, and every time 5 min.Chemoluminescence method or enzyme process is subsequently used to develop the color.The expression of YAP albumen is detected with β-actin as internal reference.

Wherein, for detecting that the one of YAP albumen resists for YAP (63.7) (Santa Cruz Biotechnology companies, product Catalog number (Cat.No.) is SC-101199)；Two resist for Anti-IgG (H+L chain) (mouse) pHb-HRP (MBL companies, catalogues Number for 458).For detecting that the one of β-actin resists for mouse anti human β-Actin monoclonal antibodies that (company of Zhong Shan Golden Bridge, catalog number is TA-09)；Two resist for Anti-IgG (H+L chain) (mouse) pHb-HRP (458) MBL companies, catalog number are.

(3) result

As a result it is as shown in Figure 2, it is seen that in each cell obtained after step one transfection, YAP turns in protein level and without si-YAP The control of dye is compared expression and is substantially reduced.

3rd, after silence YAP, in each cancerous cell, the expression of S100A7, S100A8 and S100A9 albumen is detected

The expression for importing S100A7, S100A8 and S100A9 albumen in each cancerous cell after si-YAP is detected using western blot Situation, concrete grammar are carried out referring to step 2.

Wherein, for detecting that the one of S100A7 albumen resists for rabbit-anti people S100A7 (Psoriasin) antibody (Santa Cruz Biotechnology Products, catalog number are SC67047)；Two resist for Anti-Rabbit IgG (H+L chain)-HRP (330) MBL companies, catalog number are.For detecting that the one of S100A8 albumen resists for anti-hS100A8 that (R＆B is public Department's product, catalog number is MAB4570)；Two resist for Anti-IgG (H+L chain) (mouse) pAb-HRP (MBL is public Department, 458) catalog number is.For detecting that the one of S100A9 albumen resists for goat-anti people S100A9 (Calgranulin B) antibody (Santa Cruz Biotechnology Products, catalog number is SC-8114)；Two resist for donkey anti-goat IgG-HRP (Santa Cruz Biotechnology companies, catalog number is SC-2020).

As a result it is as shown in Figure 2, it is seen that, after the YAP in silence cancerous cell, can be obviously promoted S100A7, S100A8 and The expression of S100A9 albumen.

In embodiment 3, promotion cancerous cell, the activity of YAP can suppress the expression of S100A7, S100A8 and S100A9

First, the importing wild type YAP and S127A-YAP mutant plasmid in cancerous cell

By Lipofectin2000 transfection reagents, by YAP the and S127A-YAP mutant plasmids of the wild type with His labels It is directed respectively in cancerous cell HCC94 and NCI-H292, concrete transfection method is carried out referring to kit specification.Obtain instantaneous table Up to the cell of corresponding albumen.

Wherein, the YAP plasmids of the wild type with His labels are pcDNA4/HisMaxB-YAP1 (add gene Plasmid #18978), the S127A-YAP mutant plasmid pcDNA4/HisMaxB-YAP1-S127A (addgene with His labels Plasmid#18988), this company plasmid is recorded in " Mst2and Lats kinases regulate apoptotic function of YAP. Oka T,Mazack V,Sudol M.J Biol Chem.2008 Jul 17.():.10.1074/jbc.M804380200 PubMed 18640976 " one is literary, and the public can be obtained from applicant, is only used for repeating present invention experiment.

The YAP plasmids pcDNA4/HisMaxB-YAP1 of the wild type with His labels can express sequence 1 in sequence table The recombiant protein His-YAP that the amino acid residue end of shown protein is formed after connecting 6 His labels.With His labels S127A-YAP mutant plasmids pcDNA4/HisMaxB-YAP1-S127A can express His-S127A-YAP albumen, Compared with His-YAP albumen, difference is only that the 127th serine of sequence in sequence table 1 His-S127A-YAP albumen (S) replace with alanine (A).

It is known in the art that the 127th serine (S) of YAP albumen is sported after alanine (A), YAP master can be made In core to be positioned at, biological action is played to which advantageously.

2nd, import in cancerous cell after wild type YAP and S127A-YAP mutant plasmid His-YAP in cell, YAP, The expression detection of S127-YAP, S100A7, S100A8 and S100A9 albumen

Detected using western blot and imported in cancerous cell after wild type YAP and S127A-YAP mutant plasmid in cell His-YAP, YAP, S127-YAP, S100A7, S100A8 and S100A9 expression, concrete grammar is referring to embodiment 2 Step 22 is carried out.Using GAPDH as internal reference.Wherein, S127-YAP is silk for the 127th of YAP albumen (sequence 1) Propylhomoserin there occurs the albumen after phosphatization acid.

Wherein, for detecting that the one of His-YAP albumen resists for Anti-His-tag (MBL companies, catalog number are D291-3)； Two resist for Anti-IgG (H+L chain) (mouse) pAb-HRP (458) MBL companies, catalog number are.For detecting The one of YAP albumen resists for YAP (63.7) (Santa Cruz Biotechnology companies, catalog number is SC-101199)； Two resist for Anti-IgG (H+L chain) (mouse) pAb-HRP (458) MBL companies, catalog number are.For detecting The one of S127-YAP albumen resists for p-YAP (S127) Rabbit mAb (CST companies, catalog number are D9W2I)； Two resist for Anti-Rabbit IgG (H+L chain)-HRP that (330) MBL companies, catalog number are.For detecting S100A7 The one of albumen resists for rabbit-anti people S100A7 (Psoriasin) antibody (Santa Cruz Biotechnology Products, catalogue Number be SC67047)；Two resist for Anti-Rabbit IgG (H+L chain)-HRP that (330) MBL companies, catalog number are. For detecting that the one of S100A8 albumen resists for anti-hS100A8 (R＆B Products, catalog number are MAB4570)； Two resist for Anti-IgG (H+L chain) (mouse) pAb-HRP (458) MBL companies, catalog number are.For detecting The one of S100A9 albumen resist for goat-anti people S100A9 (Calgranulin B) antibody (Santa Cruz Biotechnology Products, Catalog number is SC-8114)；Two resist for donkey anti-goat IgG-HRP (Santa Cruz Biotechnology companies, Catalog number is SC-2020).For detecting that the one of GAPDH albumen resists for mouse anti human GAPDH monoclonal antibody (middle China fir gold Bridge company, catalog number are TA-08)；Two resist for Anti-IgG (H+L chain) (mouse) pAb-HRP (MBL companies, 458) catalog number is.

The HCC94 cells and NCI-H292 that experiment setting simultaneously does not import wild type YAP and S127A-YAP mutant plasmid is thin Born of the same parents, and the HCC94 cells and NCI-H292 cells of pcDNA4/His empty carriers are imported as control.

As a result as shown in figure 3, compared with unloaded compared with control cells (NC), cell overexpression wild type YAP and S127A-YAP After plasmid, the activity of YAP increases, and has especially transfected S127A-YAP mutant plasmids.While S100A7, S100A8 The suppression different degrees of with the expression of S100A9 albumen, has especially transfected the inhibition of S127A-YAP mutant plasmids more For obvious.Thus prove, promote the activity of YAP suppress S100A7, S100A8 and S100A9 albumen in cancerous cell Expression.The testing result of the cancerous cell of wild type YAP and S127A-YAP mutant plasmid and unloaded compared with control cells (NC) are not imported Compare basically identical, no difference of science of statistics.

YAP albumen in embodiment 4, change cellular morphology (suspension culture and non-suspension culture or adhere-wall culture) regulation and control cancerous cell Activity and S100A7, S100A8 and S100A9 expression

The present inventor is by each cancerous cell (H226Br cells, NCI-H292 cells, HCC94 cells, A431 cells With FaDu cells) suspension culture and adhere-wall culture are carried out respectively.Then YAP is detected using q-PCR and/or Western blot Activity and S100A7, S100A8 and S100A9 expression.

First, cultivate each cancerous cell

1st, suspension culture (suspension)

Take the Poly-Hema solution (formula for preparing：1.2g poly-Hema are dissolved in the ethanol of 100mL95%), 2ml solution is added in the every hole of six orifice plates, opening super-clean bench uviol lamp complete to ethanol volatilization in opening lid super-clean bench, is stood Sterilization 20 minutes it is stand-by.

Cell in good condition is taken, the resuspended counting of pancreatin digestion wild Oryza species is configured to 1 × 10⁴The cell suspension of individual/ml, to 2ml cell suspension is added in the every hole of six orifice plates that poly-Hema is coated with, culture in cell culture incubator is placed in.Incubation time sets Put three gradients：24 hours (S1), 48 hours (S2) and 72 hours (S3).

2nd, the adhere-wall culture of different cell densities

Be inoculated into after cell counting in culture dish, cell culture is carried out under conditions of cell covers with culture dish floor space 60-70%. Incubation time is 48h hours.

2nd, q-PCR detects the expression of S100A7, S100A8 and S100A9 in each cancerous cell under different training methods

(1) cell total rna extracts the preparation with cDNA

Carry out referring to 2 step one 1 (1) of embodiment.

(2) real-time quantitative PCR detection gene expression

With SYBR Green as dyestuff, GAPDH is internal reference to real-time quantitative PCR, 4 parallel, negative controls of each sample Middle cDNA templates are replaced with tri-distilled water.

Wherein, it is as follows for expanding the primer of S100A7 genes：

S100A7-F：5’-CTTCCCCAACTTCCTTAGTG-3’；

S100A7-R：5’-GTAGTCTGTGGCTATGTCTC-3’.

Primer for expanding S100A8 genes is as follows：

S100A8-F：5’-AAGGGGAATTTCCATGCCGTCTA-3’；

S100A8-R：5’-GGCTGCCACGCCCATCTTTA-3’.

Primer for expanding S100A9 genes is as follows：

S100A9-F：5’-GGCACCCAGACACCCTGAACC-3’；

S100A9-R：5’-CCTCGCCATCAGCATGATGAACT-3’.

Primer sequence for expanding internal reference GAPDH is as follows：

GAPDH-F：5’-GAGTCAACGGATTTGGTCGT-3’；

GAPDH-R：5’-GACAAGCTTCCCGTTCTCAG-3’.

Reaction system is as follows：SYBR Green PCR Master Mix 10μL；1 μ L of cDNA templates；Primer 2 μ L；Pure water Complement to 20 μ L.

PCR reactions take two-step method, 60 DEG C of annealing to adopt in the annealing extension stage with 1min, 95 DEG C of degeneration 30s, absorbance are extended Collection.Relative quantification is made using △ △ Ct methods, relative expression quantity of the genes of interest in each group cDNA is calculated, computational methods are：

Ratio=2^-△△Ct=2^{-[△Ct(sample)-△Ct(calibrator)]}

In formula, Ct values refer to that the expression of genes of interest reaches the amplification cycles number experienced during threshold value, △ Ct=Ct_{(genes of interest)}-Ct_(GAPDH).Sample refers to experimental group (genes of interest), and calibrator refers to negative control (GAPDH).

(3) result

As a result, as shown in A in Fig. 4, it is seen that compared with the cancerous cell of adhere-wall culture, can be obviously promoted in cancerous cell after cell suspension The expression of the mRNA level in-site of S100A7, S100A8 and S100A9.

3rd, YAP, S127-YAP, S100A7, S100A8 in each cancerous cell under the different training methods of Western blot detections With the expression of S100A9

Concrete operations are carried out referring to 2 step 22 of embodiment.Using GAPDH as internal reference.

Wherein, for detecting that the one of YAP albumen resists for YAP (63.7) (Santa Cruz Biotechnology companies, product Catalog number (Cat.No.) is SC-101199)；Two resist for Anti-IgG (H+L chain) (mouse) pAb-HRP (MBL companies, catalogues Number for 458).For detecting that the one of S127-YAP albumen resists for p-YAP (S127) Rabbit mAb (CST companies, product Catalog number (Cat.No.) is D9W2I)；Two resist for Anti-Rabbit IgG (H+L chain)-HRP that (330) MBL companies, catalog number are. For detecting that the one of S100A7 albumen resists for rabbit-anti people S100A7 (Psoriasin) antibody (Santa Cruz Biotechnology companies Product, catalog number are SC67047)；Two resist for Anti-Rabbit IgG (H+L chain)-HRP (MBL companies, product 330) catalog number (Cat.No.) is.For detecting that the one of S100A8 albumen resists for anti-hS100A8 (R＆B Products, catalogue Number be MAB4570)；Two resist for Anti-IgG (H+L chain) (mouse) pAb-HRP (MBL companies, catalog number is 458).For detecting that the one of S100A9 albumen resists for goat-anti people S100A9 (Calgranulin B) antibody (Santa Cruz Biotechnology Products, catalog number are SC-8114)；Two resist for donkey anti-goat IgG-HRP (Santa Cruz Biotechnology companies, catalog number are SC-2020).For detecting that the one of GAPDH albumen resists for mouse anti human GAPDH monoclonal antibodies (company of Zhong Shan Golden Bridge, catalog number are TA-08)；Two resist for Anti-IgG (H+L chain) (mouse) (458) MBL companies, catalog number are to pAb-HRP.

As a result, as shown in B in Fig. 4, it is seen that compared with the cancerous cell of adhere-wall culture, cell suspension cultures can be obviously promoted YAP eggs White phosphorylation, improves the expression of S127-YAP albumen, reduces the activity of YAP albumen.In addition, and adhere-wall culture Cancerous cell compare, the table of S100A7, S100A8 and S100A9 protein level in cancerous cell can be obviously promoted after cell suspension Reach.

The present embodiment result proves that form controllable S100A7, S100A8 of cell growth and S100A9 are in cancerous cell Expression.

The activity and the table of S100A7, S100A8 and S100A9 of YAP albumen in embodiment 5, cell density regulation and control cancerous cell Reach

By each cancerous cell (H226Br cells, NCI-H292 cells, HCC94 cells and A431 cells) of different cell densities Be inoculated into adhere-wall culture in culture dish respectively, using q-PCR and western blot detect YAP activity and S100A7, The expression of S100A8 and S100A9.

First, the cancerous cell adhere-wall culture of different cell densities

Two ways is adopted altogether, and a kind of method is continuous culture method (the continuous culture different time of low-density inoculation)；Another kind is Different cell density inoculations, then continue culture same time.

First method：First cell low-density (sparse) is inoculated in culture dish, it is continuous to cultivate, change daily fresh Culture fluid, 10 days (C10) of culture co-continuous for NCI-H292 cells one, then by renewed vaccination after cell dissociation to training In foster ware, and pass on 3 times (C10S3D)；Same HCC94 cells continuously cultivate 6 days (C6), pass on 3 times (C6S3D) afterwards； H226Br cells continuously cultivate 3 days (C3), 6 days (C6) and 10 days (C10), and cell was disappeared after 10 days by continuous culture After change, renewed vaccination is in culture dish, and passes on 1 time (C10S1D), passes on 2 times (C10S2D) and passes on 3 times (C10S3D). Wherein, inoculating cell quantity<7500 cell/cm²Culture area defines low-density or Sparse.

Second method：It is inoculated into after cell counting in culture dish, cell attachment culture 48 hours is carried out under different densities.Will Inoculating cell quantity≤7500 cell/cm²Culture area defines low density cell culture or Sparse, and cell inoculation quantity is existed 7500-10000 (not containing endpoint value) individual cell/cm²Culture area is defined as normal density culture, and cell-seeding-density exists 75000-150000/cm²Culture area is defined as high density or dense or over-confluence.

2nd, q-PCR detects the expression of S100A7, S100A8 and S100A9 in each cancerous cell under different culture densities

Concrete operations are carried out referring to 4 step 2 of embodiment.

As a result as shown in A in Fig. 5, it is seen that compare with normal density culture with the cancerous cell of low density cell culture group, High Density Cultivation State can be obviously promoted the expression of the mRNA level in-site of S100A7, S100A8 and S100A9 in cancerous cell.

3rd, YAP, S127-YAP, S100A7, S100A8 in each cancerous cell under the different culture densities of Western blot detections With the expression of S100A9

As a result as shown in B in Fig. 5, it is seen that compare with normal density culture with the cancerous cell of low density cell culture group, High Density Cultivation State can promote the phosphorylation of YAP albumen, improve the expression of S127-YAP albumen, reduce the activity of YAP albumen；Together When can be obviously promoted the expression of S100A7, S100A8 and S100A9 protein level

The present embodiment result proves that culture density controllable S100A7, S100A8 and S100A9 of cell exists during adhere-wall culture Expression in cancerous cell.

The expression of embodiment 6, the activation of Hippo paths or inhibitory effect S100A7, S100A8 and S100A9 in cancerous cell

After Hippo Pathway Activations, LATS1/2 kinases can be activated, the latter can promote YAP phosphorylations, S127-YAP expression to increase Plus；Otherwise when Hippo paths are suppressed, LATS1/2 kinase-deads reduce YAP phosphorylations, the expression of S127-YAP Level is reduced.In order to confirm whether the abduction delivering of S100A7, S100A8 and S100A9 in cancerous cell is to be led to by Hippo Road mediation, the present inventor distinguishes overexpression LATS1 in the HCC94 cells and A431 cells of normal adherent growth, And the LATS1 of silencing endogenous, then have detected the expression of S100A7, S100A8 and S100A9.

First, impacts of the overexpression LATS1 to the expression of S100A7, S100A8 and S100A9 in cancerous cell

1st, the structure of overexpression LATS1 recombinant vectors

LATS1 fragments CDS area (sequence 6) is expanded with following primers F and R, by amplified production and pCMV14 (nvitrogen, Carlsbad, CA, USA) plasmid backbone carries out double digestion to produce with restricted enzyme Kpn I and Xba1 Sticky end, 16 DEG C overnight connect afterwards；By it is 10 μ l connection products heat-shock transformed enter 50 μ l competent cells in DH5 α, coated plate (contains Amp), 37 DEG C of incubated overnight, choose monoclonal, shake bacterium, carry greatly afterwards, carry out sequence verification, verify correct recombiant plasmid life Entitled pCMV14-Lats1.

F：5’-CGGGGTACCATGAAGAGGAGTGAAAAG-3 ' (recognition sequence of the underscore part for KpnI, Thereafter 1-18 position of the sequence for sequence 6)；

R：5’-GCTCTAGAAACATATACTAGATCGCGATTT-3 ' (recognition sequence of the underscore part for Xba1, Thereafter reverse complementary sequence of the sequence for the 3369-3390 positions of sequence 6).

The structure of overexpression LATS1 recombinant vector pCMV14-Lats1 is described as：By the restriction enzyme site Kpn of pCMV14 carriers Small fragment between I and Xba1 replaces with the recombiant plasmid of LATS1 genes shown in sequence 6 in sequence table.

PCMV14-Lats1 can express LATS1-Flag fusion protein, and LATS1-Flag fusion protein is in LATS1 albumen The albumen formed after amino acid residue end connection Flag labels (Flag labels are carried for pCMV14 carriers).

2nd, overexpression LATS1 recombinant vector pCMV14-Lats1 are proceeded to in each cancerous cell

For trying cancerous cell：HCC94 cells and A431 cells.

Proceed to the overexpression LATS1 recombinant vector pCMV14-Lats1 of step 1 structure in each cancerous cell for examination respectively, specifically According to Lipofectamin^TM2000 transfection reagent boxes (U.S.'s Invitrogen Products) description is transfected.Transfection 24 After hour, by the cell 1 of transfection：3 pass on, and after cell attachment, add the screening of 0.4 mg/ml G418, until choosing Positive colony cell strain.

Experiment arranges in each cell for examination the control for proceeding to pCMV14 empty carriers simultaneously.

3rd, LATS1 gene expression amounts detection

(1) RT-PCR detections

Carry out with reference to 2 step 21 of embodiment.

Wherein, it is as follows for expanding the primer sequence of LATS1 genes：

LATS1-F：5’-CACCCTTCTTGGATACCACAGC-3’；

LATS1-R：5’-CTGATTGACTCGTATGGAGGAACA-3’.

Primer sequence for expanding internal reference GAPDH is as follows：

GAPDH-F：5’-GAGTCAACGGATTTGGTCGT-3’；

GAPDH-R：5’-GACAAGCTTCCCGTTCTCAG-3’.

As a result show, after overexpression LATS1 recombinant vector pCMV14-Lats1 are proceeded to in each cancerous cell, LATS1 in cell In rna level expression apparently higher than the control for proceeding to pCMV14 empty carriers.

(2) western blot detections

Carry out referring to 2 step 22 of embodiment.The table of LATS1-Flag fusion protein is detected using Flag tag antibodies specially Up to situation.

As a result show, after overexpression LATS1 recombinant vector pCMV14-Lats1 are proceeded to in each cancerous cell, can detect in cell To obvious LATS1-Flag fusion protein signal.

4th, detect the table of YAP, S127-YAP, S100A7, S100A8 and S100A9 in the cancerous cell of overexpression LATS1 Up to situation

To overexpression, the cancerous cell of LATS1 to be covered with cell and carry out under the density of culture dish floor space 60-70% adhere-wall culture. Incubation time is 48h.

The cell of different disposal is carried out YAP in the cancerous cell of western blot detection overexpression LATS1, S127-YAP, The expression of S100A7, S100A8 and S100A9.Western blot concrete operations are carried out referring to 2 step 22 of embodiment. Using GAPDH as internal reference.

As a result it is as shown in Figure 6：After overexpression LATS1, S127-YAP levels are improved, while S100A7, S100A8 and S100A9 Expression also significantly improve (A and B in Fig. 6).

2nd, impact of silence LATS1 to the expression of S100A7, S100A8 and S100A9 in cancerous cell

1st, the endogenous LATS1 genes of si-LATS1 silenced cells are proceeded to in each cancerous cell

At 700,000 cancerous cell of 60mm culture dishs middle berth (HCC94 cells or A431 cells), 24h is cultivated.By transfection examination Agent (jetPRIME), Buffer and the siRNA for disturbing YAP genes (sequence 2) to express are mixed, standing 10min, Uniformly it is added drop-wise in culture medium.Cell is received after 48h standby.

Wherein, for disturbing the siRNA that LATS1 genes (sequence 6) are expressed to be that two by shown in following C and D are single-stranded The double-strand of annealing.

C：5 '-GCAGCGUCUACAUCGUAAATT-3 ' (sequence 7)；

D：5 '-UUUACGAUGUAGACGCUGCTT-3 ' (sequence 8).

Experiment is arranged without the corresponding cell of si-LATS1 transfections simultaneously as control.

2nd, LATS1 gene expression amounts detection

(1) RT-PCR detections

Operation is with step one.

As a result show, proceed to after si-LATS1 in each cancerous cell, in cell, LATS1 is significantly lower than in rna level expression The control of si-LATS1 is not proceeded to.

(2) western blot detections

Operation is with step one.

As a result show, proceed to after si-LATS1 in each cancerous cell, in cell, LATS1 is significantly lower than in protein expression amount The control of si-LATS1 is not proceeded to.

3rd, YAP, S127-YAP, S100A7, S100A8 and S100A9 in the cancerous cell of western blot detections silence LATS1 Expression

To silence, the training method of the cancerous cell of LATS1 arranges as follows：Suspension culture；High density adhere-wall culture (75000-150000/cm²Culture area).Incubation time is 48h.

The cell of different disposal is carried out YAP in the cancerous cell of western blot detection silences LATS1, S127-YAP, The expression of S100A7, S100A8 and S100A9, concrete operations are with step one.

As a result it is as shown in Figure 6：Compared with the control (si-NC) for not proceeding to si-LATS1, the LATS1 expression of silencing endogenous The phosphorylation and S100A7, S100A8 and S100A9 of YAP can substantially be suppressed in the adherent thin of suspension cell and High Density Cultivation Expression (C and D in Fig. 6) in born of the same parents.

The present embodiment result shows that the either expression of silencing endogenous LATS1, or external source imports equal controllables YAP of LATS1 Phosphorylation level and S100A7, S100A8 and S100A9 expression.

The expression of embodiment 7, internal microenvironment inducible S100A7, S100A8 and S100A9 in lotus knurl tissue

The present inventor by each cancerous cell (NCI-H292 cells, HCC94 cells, A431 cells, FaDu cells and H226Br cells) nude mice by subcutaneous is inoculated into respectively carries out lotus knurl.Then S100A7, S100A8 during SABC detection lotus knurl is organized With the expression of S100A9.It is specific as follows：

First, cancer cell inoculation nude mice is carried out into lotus knurl

Pancreatin digestion is collected by centrifugation cell after culture medium is resuspended, uses physiology in exponential phase, each cancerous cell in good condition Saline (0.9%NaCl solution) carries out cell counting after cleaning three times；Most cell preparation becomes 5 × 10 at last⁷The cell of/mL hangs Liquid；It is each to 5 week old Female nude mice (coming from Beijing Vital River Experimental Animals Technology Co., Ltd.) forelimb oxter Bilateral injections 100 μ L, raise in SPF level environment (the 3rd Affiliated Hospital's Animal House of Department Of Medicine, Peking University), observe which into tumor situation.

2nd, the same step of ImmunohistochemistryMethods Methods.

Nude mice is put to death when mouse-borne tumor diameter reaches 0.5-1cm, tumor is removed and is fixed stand-by in formalin.According to as follows Carry out SABC：

1st, the preparation of paraffin section

The good piece of tissue of formalin fix is taken, dimethylbenzene is transparent after graded ethanol dehydration, immersed in the paraffin of dissolving, waxdip three times. Subsequently tissue and dewaxing are poured in embedded box, wax stone are removed after solidification to be cooled, are put to paraffin slicing machine section, slice thick 5 μm of degree, section are dragged for adhesion microscope slide after spreading out piece in being put into 42 DEG C of hot water, are dried standby in 70 DEG C of baking ovens.

2nd, histogenic immunity group

Take paraffin section and be put in 70 DEG C of baking ovens and dry 1-2 hours, gradient alcohol aquation after dimethylbenzene dewaxing.Subsequently by slice, thin piece and lemon Lemon acid antigen retrieval buffers are put into pressure cooker mesohigh heating 1min 40s and carry out antigen retrieval.Then 3% (volume fraction) peroxide Change hydrogen-PBS incubation at room temperature 15min fire extinguishing Endogenous peroxidases, PBS is washed once afterwards with 3% (volume fraction) Ox blood serum-PBS Room temperature closes 20min, and subsequently incubation one resists overnight or 37 DEG C of 1h, PBS to wash 3 times (each 10min), is incubated HRP labellings Two resist, 15min room temperatures, PBS are washed 3 times (each 5min), DAB colour developing 3min after haematoxylin redye, wash, ladder Degree ethanol dehydration, the transparent rear mounting of dimethylbenzene, basis of microscopic observation staining conditions ".

As a result it is as shown in Figure 7, it is seen that compared with the cell (see embodiment 1) of In vitro culture, except the lotus knurl group of NCI-H292 Knit outer, the positive cell number showed increased of S100A7, S100A8 and S100A9 in the lotus knurl tissue of other cancerous cell, especially It is especially pronounced during the lotus knurl of H226Br and HCC94 cells is organized.As can be seen here internal microenvironment also can induce S100A7, S100A8 and S100A9 expression in vivo.

Claims

1. application of the material of YAP protein actives in following (a) or (b) can be suppressed：

B () is prepared for promoting S100A7 albumen and/or S100A8 albumen and/or S100A9 albumen tables in cancerous cell The product for reaching.

2. it is a kind of to be used to promote S100A7 albumen and/or S100A8 albumen and/or S100A9 protein expressions in cancerous cell Product, its active component is the material that can suppress YAP protein actives.

3. any one application in following (c) or (d) in following (1)-material shown in (5)：

(1) YAP albumen；

(2) S127A-YAP albumen；The S127A-YAP albumen is by the 127th silk of the YAP albumen Histidine mutations are the albumen of gained after alanine；

(3) encoding gene of the YAP albumen, or expression cassette, the weight of the encoding gene containing the YAP albumen Group carrier or transgenic cell；

(4) encoding gene of the S127A-YAP albumen, or the coding base containing the S127A-YAP albumen The expression cassette of cause, recombinant vector or transgenic cell；

(5) material of YAP protein actives can be strengthened；

D () is prepared for S100A7 albumen in anticancer and/or S100A8 albumen and/or S100A9 albumen tables The product for reaching.

4. a kind of for S100A7 albumen and/or S100A8 albumen and/or S100A9 protein expressions in anticancer Product, its active component is any one in following (1)-material shown in (5)：

(1) YAP albumen；

(5) material of YAP protein actives can be strengthened.

5. according to arbitrary described application or product in claim 1-4, it is characterised in that：It is described to suppress YAP The material of protein active is any one in following (a1) and (a2)：

(a2) material of YAP protein phosphorylations can be promoted；

The material that YAP protein actives can be strengthened is the material that can suppress YAP protein phosphorylations.

6. application according to claim 5 or product, it is characterised in that：The silence YAP albumen of being capable of The nucleic acid molecules of encoding gene be two single-chain nucleic acids shown in sequence in sequence table 3 and sequence 4 are annealed to be formed it is double Chain nucleic acid；And/or

The material that YAP protein phosphorylations can be promoted in following any one：LATS1 albumen, coding are described The encoding gene of LATS1 albumen, the expression cassette of the encoding gene containing the LATS1 albumen, recombinant vector or turns Gene cell, LATS1 protein active accelerator；And/or

The material that YAP protein phosphorylations can be suppressed is LATS1 protein active inhibitor；

Specifically, the Lats1 protein actives inhibitor is the nucleic acid for being capable of the encoding gene of Lats1 albumen described in silence Molecule；

More specific, described " being capable of the nucleic acid molecules of the encoding gene of Lats1 albumen described in silence " is by sequence In table, sequence 7 and two single-chain nucleic acids shown in sequence 8 are annealed the double-strandednucleic acid to be formed.

7. method I or method II, it is characterised in that：

Methods described I is S100A7 albumen and/or S100A8 albumen and/or S100A9 albumen tables in promotion cancerous cell The method for reaching, comprises the steps：Suppress YAP protein actives in the cancerous cell, so as to realize promoting the cancer thin The expression of S100A7 albumen and/or S100A8 albumen and/or S100A9 albumen in born of the same parents；

In methods described I, YAP protein actives described in the cancerous cell are suppressed to be by following (b1)-(b3) At least one of realize：

(b1) thing that can suppress YAP protein actives in the cancerous cell described in importing claim 5 or 6 Matter；

(b2) cancerous cell described in culture in the way of suspension culture；

(b3) cancerous cell described in culture in the way of adhere-wall culture, and the culture density of the cancerous cell is 75000-150000 cell/cm²Culture area；

Methods described II is S100A7 albumen and/or S100A8 albumen and/or S100A9 albumen table in anticancer The method for reaching, comprises the steps (A) or (B) or (C), so as to realize suppressing S100A7 in the cancerous cell The expression of albumen and/or S100A8 albumen and/or S100A9 albumen：

(A) encoding gene of YAP albumen is imported in the cancerous cell, YAP albumen in the cancerous cell is improved Expression；

(B) encoding gene of S127A-YAP albumen is imported in the cancerous cell, makes the cancerous cell expression described S127A-YAP albumen；The S127A-YAP albumen is by the 127th mutant serine of the YAP albumen For the albumen of gained after alanine；

(C) YAP protein actives in the cancerous cell are strengthened；

In (C), it is by leading in the cancerous cell to strengthen YAP protein actives described in the cancerous cell Enter the material that can strengthen YAP protein actives described in claim 5 or 6 to realize.

8. according to arbitrary described application or product or method in claim 1-7, it is characterised in that：The YAP In the aminoacid sequence such as sequence table of albumen shown in sequence 1；The nucleotide sequence of the encoding gene of the YAP albumen is such as In sequence table shown in sequence 2；And/or

127th serine of sequence in sequence table 1 is such as replaced by the aminoacid sequence of the S127A-YAP albumen Obtained by after alanine shown in sequence；The nucleotide sequence of the encoding gene of the S127A-YAP albumen is such as by sequence In table the oligonucleotide " TCC " of the 379-381 positions of sequence 2 replace with " GCC " afterwards gained sequence shown in；

In the aminoacid sequence such as sequence table of the LATS1 albumen shown in sequence 5；The coding of the LATS1 albumen In the nucleotide sequence such as sequence table of gene shown in sequence 6；And/or

In the aminoacid sequence such as sequence table of the S100A7 albumen shown in sequence 9；And/or

In the aminoacid sequence such as sequence table of the S100A8 albumen shown in sequence 10；And/or

In the aminoacid sequence such as sequence table of the S100A9 albumen shown in sequence 11；And/or

Phosphorylation of the YAP albumen phosphatization acid for the 127th serine of protein shown in sequence 1 in sequence table.

9. according to arbitrary described application or product or method in claim 1-8, it is characterised in that：The cancerous cell For in following any one：Lung carcinoma cell, squamous carcinoma of the cervix cell, epidermis cancerous cell and Dental clinic；

Specifically, the lung carcinoma cell is NCI-H292 cells or H226Br cells；The squamous carcinoma of the cervix cell is HCC94 cells；The epidermis cancerous cell is A431 cells；The Dental clinic is FaDu cells.

10. methods described I in claim 7 is preparing S100A7 albumen and/or S100A8 albumen and/or S100A9 Application in albumen.