CN104017868A

CN104017868A - Application of SETD4 to preparation of pancreatic cancer diagnosis and / or prognosis kit and application of SETD4 blocker to preparation of medicament for treating pancreas cancer

Info

Publication number: CN104017868A
Application number: CN201410227551.1A
Authority: CN
Inventors: 李晨蔚
Original assignee: Jiang Suxin Noboru Bioisystech Co Ltd
Current assignee: Shanghai still Biotechnology Co., Ltd.
Priority date: 2014-05-27
Filing date: 2014-05-27
Publication date: 2014-09-03
Anticipated expiration: 2034-05-27
Also published as: CN104017868B

Abstract

The invention provides application of SETD4 to preparation of pancreatic cancer diagnosis and / or prognosis kit and application of an SETD4 blocker to preparation of medicaments for treating pancreas cancer. Through studies, the invention confirms that SETD4 has high expression in pancreatic tumor cells and tumor stem cells, and can be used in the diagnosis of pancreatic cancer, and used as therapeutic effect prediction index for predicting prognosis; blocking of SETD4 expression can inhibit pancreatic tumor growth and pancreatic cancer stem cells, and can be used as a novel medicament for pancreatic cancer treatment and a novel target for biological immunotherapy.

Description

Purposes and the SETD4 blocker purposes in preparation treatment carcinoma of the pancreas medicine of SETD4 in preparation diagnosis of pancreatic cancer and/or prognosis kit

Technical field

The present invention relates to purposes and the SETD4 blocker purposes in preparation treatment carcinoma of the pancreas medicine of ZNFN3A1 SETD4 in preparation diagnosis of pancreatic cancer and/or prognosis kit, belong to medical biotechnology field.

Background technology

Ductal adenocarcinoma of pancreas (PDAC) is one of malignant tumour that current grade malignancy is the highest, is also one of main lethal cancer in the whole world, estimates to cause every year 227,000 deaths.Conventionally developed into late period when diagnosis, only can excision lower than 20% carcinoma of the pancreas; 40% presents part invades profit, cannot excision focus; When remaining diagnosing patient, occur that focus shifts.And carcinoma of the pancreas is to conventional chemotherapy, radiotherapy insensitive (1,2).Current treatment plan cannot obviously extend patient's life, and does not significantly improve in the past 35 years of clinical treatment result, and overall 5 years survival rates are in 5% left and right, and most of patients with terminal mean survival time (MST) was less than 1 year (2,3).The early stage transfer of carcinoma of the pancreas is its main fatal reason.Evidence suggests, this process may realize by tumor stem cell CSCs, and this class cell has the characteristic (4) of Epithelial and stromal conversion (EMT).

Tumour be formed with two kinds of theories.Stochastic theory thinks that each tumour cell is homogeneity, and each tumour cell has the possibility that forms a new tumour.But its multiple fission cycle that enters cell is the random occurrence control by some small probabilities.Hierarchy theory is thought and is had a cell subsets in tumour cell: tumour promotion cell, and only have the cell in this subgroup just to have the ability that forms new tumour, this type of cell has the ability of self and Multidirectional Differentiation simultaneously.Tumour promotion cell, tumor stem cell has caused generation and the development (13) of tumour.

AACR(11 in 2006) (American Association for Cancer Research) to the definition of tumor stem cell be: in tumour, have self-renewal capacity and can produce the cell of heterogeneous tumour cell, this cell can be divided into the different cells that form tumour.Normal stem cell has similar biological characteristics to tumor stem cell: self, infinite multiplication, Multidirectional Differentiation etc., and difference is that the unlimited multiplication capacity of normal stem cell is subject to Adjust System, can make the multiplication capacity of normal stem cell out of control by sudden change; And the propagation of tumor stem cell is not modulated.Stem cell in vivo the survival time long, there is the potential of self-replacation, more likely accumulation sudden change, thereby become tumor stem cell (12) by quantitative change to qualitative change.

Tumor stem cell mainly utilizes cell surface marker to define at present, and each tumour cell has unique cell surface marker can be identified.The Dick of University of Toronto in 1997 and Bonnet etc. isolate leukemia tumor stem cell (CD 34+/CD38-) (6) first.Li (Li Chenwei), Clarke in 2007 and Simeone etc. confirm that carcinoma of the pancreas stem cell exists (CD44+CD24+ESA+) first, and such cell has high tumorigenesis ability and can self and produce the tumor stem cell characteristic (7) of different cell colonys.The report CD133 positive cells such as Hermann have height tumorigenicity and resistance (8).The research of Rasheed etc. finds that ALDH positive tumor cell has the characteristic of tumor stem cell and the characteristic of EMT (9).Li (Li Chenwei) and Simeone report c-Met are a kind of new carcinoma of the pancreas stem cell surface marks, and can be used as the Effective target site (5) for the treatment of.Rhim etc. are verified enters sanguimotor tumour cell and compares CD44+CD24+cell with tumor in situ cell and increased by 100 times (18).

Conventional tumor stem cell sorting method has 3 kinds.1, by phenotype sorting carcinoma of the pancreas stem cell: tumor stem cell and non-tumor stem cell do not have difference on morphology.A kind of method of distinguishing tumor stem cell and non-tumor stem cell is to pass through surface marker.Li etc. (7) are by heteroplastic transplantation model cultivator carcinoma of the pancreas primary cell, go out the high tumorigenicity cell mass of approximately 0.2% ~ 0.8% phenotype as CD44+CD24+ESA+ taking CD44, CD24, ESA as surface marker by selected by flow cytometry apoptosis, and become in knurl model to confirm that this kind of cell has high tumorigenesis ability animal.Huang etc. (14) sub-elect the high tumorigenicity cell of CD44+CD24+ in pancreas cancer cell strain PANC-1 taking CD44, CD24 as surface marker, and in the body of nude mice, become the tumorigenesis ability of the cell that confirms this phenotype in knurl experiment strong 20 times compared with the cell of CD44-CD24-.Hermann etc. (8) sub-elect the cell of CD133+ at Tissues of Human Adenocarcinoma of Pancreas and pancreas cancer cell strain L3.6pl by immunological magnetic bead sorting method, and in tumorigenesis experiment, confirming that this kind of cell has high tumorigenesis ability in the body of athymic mouse, 500 this kind of cells can become knurl.2, side population cell sorting: in the time there is no the surface markers of clear and definite tumor stem cell, can utilize characteristic that side population cell (side population, SP) pumps Hoechest dyestuff in conjunction with Flow Cytometry sorting SP cell.Zhou etc. (15), with Hoechst 33342 and PI dyes PANC-1 cell, then sub-elect side population cell by Flow Cytometry.Confirm to have the light SP cell dying in PANC-1 cell strain with fluorescent microscope morphological examination.3, sacculus is cultivated: suspension cell sacculus culture method is another kind of common stem cell sorting method.Gou etc. (16) are taking DMEM-F12 as basic medium.Add the factor such as EGF, Regular Insulin, in PANC-1 cell strain, turned out sacculus cell, dyeed and find that 98.10% ± 1.26% cell is not painted by Hoeehst.In the body of nude mice, become knurl experiment also to confirm that sacculus cell is compared with strong approximately 20 times of the one-tenth knurl ability of attached cell.

Initial is Su (var) 39 at 3 regulatory factors of fruit bat, the shuttle base end of Enhancer of zeste (E (z)) and trithorax (trx) is all found a common sequence motifs being made up of 130 left and right amino acid, therefore get its prefix letter by this sequence motifs called after SET structural domain (17).SET structural domain is the structural domain of a high conservative, and in eukaryote, the formation of the wide participation activation of SET structural domain and inhibition complex body, plays an important role to the generation development of organism.Find at present the albumen that contains SET structural domain of isolation identification and theory hypothesis of kind more than 300, be referred to as SET gene family.

The aminoterminal variation of histone is modified and has been formed a complicated network, has greatly enriched the information content of traditional genetic code.The modification that methylates of histone is mainly that the ZNFN3A1 that contains SET structural domain by a class is carried out, but the protein that not contains SET structural domain is all the histone that methylates, have been found that at present and confirm the ZNFN3A1 of general kind more than 700 containing SET structural domain, wherein people source just has a kind more than 100, and they have participated in the different physiological roles (18) such as heterochromatin formation, Genomic Imprinting, X x chromosome inactivation and transcriptional control.Histone methylated abnormal and multiple human diseases is as relevant in the generation of tumour etc.Now the most of member of clear and definite SET gene family has the function of ZNFN3A1.Because the member of SET gene family is more, study at present and more be:

(1) SU (VAR) 3-9 subfamily: SU (VAR) 3-9 subfamily has more than 30 member, mainly comprises the albumen such as Suv39h1, Suv39h2, G9a, ESET, EuHMTaseI.Experiment shows that, after the dual-gene Suv39h1/h2 that knocks out mouse, H3K9 methylation level reduces, the obvious deficient in stability of genome; Lymph node puncture confirms have 33% mouse to have B cell lymphoma.Think at present, suv39h1/h2 is by interacting and regulate Cyeline E with Rb, the factor that therefore any destruction Suv39h1/h2 is combined with Rb all play a significant role in tumour generation (19).

(2) SET 1 subfamily: SET 1 subfamily comprises E (z) subfamily, be poly-comb albumen, Enhancer of Zeste1 and 2 (EZH1, EZH2), and mankind Trithorax albumen MLL1-3 and Tritho-rax associated protein (ALR).This family member's common trait is that the downstream of next-door neighbour SET structural domain exists a cysteine rich domain CXC.Due to SET 1 family gene control cell fission, differentiation and growth, therefore the isogenic imbalance of PcG, trxG can cause the generation of tumour.Experiment has confirmed that trxG subfamily member and tumour are formed with close relationship, as: band normal producer in leukemia people in MLL1 designation of chromosome 11q23 district is reset, swivel base, MLL1 gene is ruptured, expression deletion SET structural domain, lose the function of MLL1 gene itself, or there is partial replication, disappearance, form fusion rotein and obtain new function (20).In about 80% dissimilar leukemia children, also there is this type of sudden change.

(3) SET 2 subfamilies: SET2 subfamily comprises SET2, NSD (nuclear receptor-binding SET-domain-containing).SET2 has the activity of H3K36 methyltransgerase.Because NSD comprises SET structural domain, with ySET2 height homology, infer that NSD albumen also has the activity of methyltransgerase, participates in chromosomal adjusting (21).In the time that NSD2 crosses expression, can Induction Transformation.IgH chain as visible in myelomatosis patient and NSD2 merge, and cause the undue expression of H chain.Can promote development when NSD2 low expression level as can be seen here, high level expression or express imbalance and cause malignant transformation of cells, causes tumour to form.

(4) RIZ subfamily: RIZ be Rb in conjunction with albumen, be also the cofactor of estrogen receptor.Having SET structural domain at aminoterminal, there is another conservative structural domain in shuttle cardinal extremity, therefore can be by Dimerized.The minimizing of common RIZ1 gene and disappearance in many tumours, the high expression level of RIZ2, prompting RIZ has special negative and positive regulating effect.This external 37% primary tumo(u)r is as visible RIZ gene SET structural domain district in colon, stomach, carcinoma of the pancreas or the generation phase shift mutation of contiguous SET structural domain district and missense mutation, make 106 halfcystines in SET structural domain change Threonine into, lose the activity of methyltransgerase, cause that cell proliferation and tumour form.RIZ gene family has been used to the target of clinical cancer therapy at present.

SETD4(ZNFN3A1) belong to the newcomer in SET family protein, at present about the rare bibliographical information of functional study of SETD4.SETD4 is mainly made up of two structural domains, is positioned at the SET structural domain (people's the 34th to 279 amino acids) of C-terminal and is positioned at the Binding Capacity structural domain (the 307th of people to 425 amino acid) of aminoterminal end.SET structural domain is by the SET-N at two ends, SET-C and middle SET-I composition, SET-N and SET-C high conservative, carboxyl terminal comprises many direct and active relevant amino-acid residues, the insertion number of SET-I is the amount doesn't matter, substrate and the cofactor of energy specific recognition methyltransgerase.

Research finds that blocking-up SETD4 increases the susceptibility of HepG2 liver cancer cell to Xarelto.The phosphorylation of AKT is lowered in Xarelto and blocking-up SETD4 combination therapy, thus the death of induction liver cancer cell.The susceptibility biomarker of possible Xarelto and the novel method (22) of liver cancer patient Xarelto combination therapy have been found in this research.Another research finds that the generation of ZNFN3A1 SETD4 and mammary cancer is relevant.In several breast cancer cell lines, the expression of quantitative PCR detection SETD4 raises.Western confirms the high expression level of SETD4.Significantly suppress its propagation and postponed G1/S cell cycle phase transition and do not affect apoptosis at breast cancer cell line blocking-up SETD4.In addition, Western data presentation blocking-up SETD4 reduces the expression of cyclin D1, and prompting SETD4 participates in the regulation and control of cell cycle.Therefore, SETD4 is in the vital effect of playing of mammary cancer, and may become the method (23) of diagnosis and the treatment of new mammary cancer.

At present not yet there is SETD4 to be applied in the report in pancreatic neoplasm.

reference:

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2. Stathis,A.,and Moore,M.J. Advanced pancreatic carcinoma:current treatment and future challenges. Nat.Rev.Clin.Oncol. 2010. 7,163–172.

3. Siegel R, Naishadham D, Jemal A. Cancer statistics, 2013. CA Cancer J Clin. Jan 2013;63(1):11–30.

4. Li Y, Kong D, Ahmad A, Bao B, Sarkar FH. Pancreatic cancer stem cells: Emerging target for designing novel therapy. Cancer Lett. 2012;338(1):94–100.

5. Li C, Wu JJ, Hynes M, Simeone DM et al. c-Met is a marker of pancreatic cancer stem cells and therapeutic target. Gastroenterology. 2011;141(6): 2218–2227

6. Bonnet D, Dick JE. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med 1997;3:730–737.

7. Li C, Heidt DG, Dalerba P, Simeone DM，et al Identification of pancreatic cancer stem cells. Cancer Res 2007;67:1030–1037.

8. Hermann PC, Huber SL, Herrler T, et al. Distinct populations of cancer stem cells determine tumor growth and metastatic activity in human pancreatic cancer. Cell Stem Cell. 2007;1(3):313–323.

9. Rasheed ZA, Yang J, Wang Q, et al. Prognostic significance of tumorigenic cells with mesenchymal features in pancreatic adenocarcinoma. J Natl Cancer Inst. 2010;102(5):340–351.

10. Rhim AD, Mirek ET, Aiello NM, et al. EMT and dissemination precede pancreatic tumor formation. Cell. 2012;148(1–2):349–361

11．Clarke MF, Dick JE, Dirks PB, Eaves CJ, Jamieson CH, Jones DL, Visvader J, Weissman IL, Wahl GM. Cancer stem cells--perspectives on current status and future directions: AACR Workshop on cancer stem cells. Cancer Res 2006 Oct 1; 66(19): 9339-9344.

12．Dalerba P, Cho RW, Clarke MF. Cancer stem cells: models and concepts. Annu Rev Med 2007; 58: 267-284.

13．Reya T, Morrison SJ, Clarke MF, Weissman IL. Stem cells, cancer, and cancer stem cells. Nature 2001 Nov 1; 414(6859): 105-111.

14．Huang P, Wang CY, Gou SM, Wu HS, Liu T, Xiong JX. Isolation and biological analysis of tumor stem cells from pancreatic adenocarcinoma. World J Gastroenterol 2008 Jun 28; 14(24): 3903-3907.

15．Zhou J, Wang CY, Liu T, Wu B, Zhou F, Xiong JX, Wu HS, Tao J, Zhao G, Yang M, Gou SM. Persistence of side population cells with high drug efflux capacity in pancreatic cancer. World J Gastroenterol 2008 Feb 14; 14(6): 925-930.

16．Gou S, Liu T, Wang C, Yin T, Li K, Yang M, Zhou J. Establishment of clonal colony-forming assay for propagation of pancreatic cancer cells with stem cell properties. Pancreas 2007 May; 34(4): 429-435.

17. Qian C , Zhou MM .SET domain protein lysine methyltransferases: Structure, specificity and catalysis. Cellular and molecular life sciences : CMLS 2006 Dec; 63 (23 ):2755-63

18. Strahl BD , Allis CD .The language of covalent histone modifications.Nature 2000 Jan; 403 (6765 ):41-5.

19. Peters AH , O'Carroll D , Scherthan H , Sibilia M ,Jenuwein T .Loss of the Suv39h histone methyltransferases impairs mammalian heterochromatin and genome stability.Cell 2001 Nov; 107 (3 ):323-37.

20. Dou Y , Hess JL .Mechanisms of transcriptional regulation by MLL and its disruption in acute leukemia.International journal of hematology 2008 Jan; 87 (1 ):10-8

21. Strahl BD , Grant PA , Briggs SD , , Hunt DF , Allis CD Set2 is a nucleosomal histone H3-selective methyltransferase that mediates transcriptional repression. Mol Cell Biol 2002 Mar;22 (5): 1298-306

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23. Faria JA, Corrêa NC, de Andrade C, Rodrigues TS, de Goes AM, Gomes DA, Silva FP. SET domain-containing Protein 4 (SETD4) is a Newly Identified Cytosolic and Nuclear Lysine Methyltransferase involved in Breast Cancer Cell Proliferation. J Cancer Sci Ther. 2013 Jan 21;5(2):58-65。

Summary of the invention

The object of the invention is to make up the blank of prior art, the purposes of SETD4 in preparation diagnosis of pancreatic cancer and/or prognosis kit is provided.The purposes of SETD4 blocker in preparation treatment carcinoma of the pancreas medicine is also provided.

First confirm that SETD4 is at pancreatic tumor cell, high expression level in pancreas diagnosis of hepatic metastases cell and tumor stem cell.Further confirm the relation of SETD4 in the positive level of pancreatic tumor cell and prognosis.Find that SETD4 quantitative PCR and immunohistochemical methods inspection can be used to diagnosis of pancreatic cancer, and as the index prediction prognosis of predicted treatment effect.

The conventional treatment of cancer at present, as radiotherapy, chemotherapy can be killed the major part malignant cell rapidly that grows really, but but act on very micro-ly to being present in the tumor stem cell of the quantity rareness in tumour, the propagation of remaining tumor stem cell after treatment, is enough to impel cancer return and transfer.Effectively oncotherapy should comprise the treatment plan for tumor stem cell group.Tumor stem cell is self ad infinitum, and has relatively static feature, and these characteristics and Epithelial and stromal Transformed E MT combine, and just can make them avoid conventional chemotherapy method inducing cell death.Lead oncogenic transfer, for causing tumour patient main causes of death.

For finding new pancreatic tumour method and target spot, a series of experimental study and the clear and definite effect of SETD4 in treatment of pancreatic cancer are carried out.First experiment finds that blocking-up SETD4 can suppress the growth of pancreatic tumor cell.Experiment in vitro utilizes Panc-1 cell strain to carry out MTT analysis, shows that ShRNA blocking-up SETD4 can effectively suppress the growth of pancreatic neoplasm.Inoculate Panc-1 cell simultaneously and set up people source carcinoma of the pancreas animal model to immunodeficient mouse and study, experimental results show that blocking-up SETD4 can suppress pancreatic neoplasm growth in body.Compare with common Panc-1 cell, the tumor growth that after Panc-1 cell transfecting SETD4 ShRNA, subcutaneous injection nude mice forms is suppressed.And blocking-up SETD4 associating carcinoma of the pancreas choice drug gemcitabine (Gemcitabine) result for the treatment of is better.Utilize the experimental result of primary people source carcinoma of the pancreas animal model (Patient-Derived Xenograft-PDX) and utilize the result of Panc-1 to coincide.

Can suppress the mechanism of the growth of pancreatic tumor cell for further studying blocking-up SETD4, the tumor tissues that experimentation on animals is obtained produces unicellular through collagen protein enzymic digestion, first utilize CD44 to separate with C-met antibodies streaming and obtain carcinoma of the pancreas stem cell, the impact of research blocking-up SETD4 on tumor stem cell.Carry out the impact of quantitative PCR analysis research blocking-up SETD4 on EMT path simultaneously.Above-mentioned experiment finds that SETD4 ShRNA can suppress pancreatic neoplasm stem cell significantly; Quantitative PCR detection finds that blocking-up SETD4 can suppress the EMT of Snail mediation, thereby reaches result for the treatment of.

SETD4 can be used as the novel targets for the treatment of of pancreatic cancer new drug and biological immune treatment.

The present invention is by studies confirm that SETD4 high expression level in pancreatic tumor cell and tumor stem cell, and SETD4 can be applicable to diagnosis of pancreatic cancer, and as the index prediction prognosis of predicted treatment effect; Blocking-up SETD4 expresses and can suppress pancreatic neoplasm growth, suppresses pancreatic neoplasm stem cell, can be used as the novel targets for the treatment of of pancreatic cancer new drug and biological immune treatment.

Brief description of the drawings

Fig. 1 is qRT-PCR contrast normal pancreatic tissue, pancreatitis, and in pancreatic tumor tissue and pancreatic neoplasm stem cell, the expression level of SETD4 changes comparison diagram.

Fig. 2 is SETD4 expression level and the Prognostic significance figure of 21 routine Patients with Pancreatic Cancers.

Fig. 3 is the impact of experiment in vitro inspection blocking-up SETD4 on pancreatic cancer cell survival rate.

Fig. 4 is that experimentation on animals inspection suppresses the impact of SETD4 on pancreatic cancer growth.

Fig. 5 is the impact of experiment in vitro inspection blocking-up SETD4 on pancreatic cancer cell EMT.

Embodiment

The immunohistochemical experiment, qRT-PCR, MTT, the foundation of carcinoma of the pancreas transplanted tumor animal model and the operation steps of curative effect testing method, carcinoma of the pancreas stem cell sorting method and statistical analysis method that in the present invention, relate to are as follows respectively.

one, immunohistochemical methods operation steps

1, dewaxing and aquation: before dewaxing, organization chip should be placed at room temperature 60 minutes or 60 DEG C of thermostat containers in toast 20 minutes.

A) organization chip is placed in dimethylbenzene and soaks 10 minutes, is soaking 10 minutes after changing dimethylbenzene

B) in dehydrated alcohol, soak five minutes

C) in 95% ethanol, soak five minutes

D) in 75% ethanol, soak five minutes

2, antigen hot repair is multiple:

Microwave-oven-heating heats 0.01 sodium citrate buffer (ph6.0) in microwave oven to be put into organization chip to boiling, power-off, interval 5-10 minute, 1-2 time repeatedly.For the fixing paraffin embedded tissue chip of formalin

3, immunohistochemical staining SP method concrete operation step:

(1) dewaxing, aquation

(2) PBS wash 2-3 time each 5 minutes

(3) 3%H2O2 (80% methyl alcohol) drips on TMA, and room temperature leaves standstill 10 minutes

(4) PBS wash 2-3 time each 5 minutes

(5) antigen retrieval

(6) PBS wash 2-3 time each 5 minutes

(7) drip normal goats serum confining liquid, room temperature 20 minutes, gets rid of unnecessary liquid

(8) drip the anti-50 μ l of Ι, room temperature leaves standstill 1-2 hour (antibody: SETD4 antibody (N-20) Santa Cruz Biotech)

(9) 4 DEG C need be 37 DEG C of rewarmings 45 minutes after spending the night

(10) PBS wash 3 times each 2 minutes

(11) drip II anti-45-50 μ l, room temperature leave standstill or 37 DEG C 1 hour

(12) II can add 0.05% tween-20. in resisting

(13) PBS wash 3 times each 5 minutes

(14) DAB colour developing 5-10 minute, grasps dye levels under the microscope

(15) PBS or tap water rinse 10 minutes

(16) haematoxylin redyeing 2 minutes, hydrochloride alcohol differentiation

(17) tap water rinses 10-15 minute

(18) dehydration, transparent, mounting, microscopy.

4, result is passed judgment on

Cancerous tissue and healthy tissues to tissue sample are evaluated respectively, occur the mark of brown yellow granule as positive expression using cell.Work out the following judgment criteria of expressing according to reference: dyeing area <10%-0 divides; >11% <25%-1 divides; >26% < 50%-2 divides; >51%-3 divides.Staining power feminine gender-0 point; Weak-1 point; In-2 points; By force-3 points.

two, qRT-PCR operation steps

One) total RNA extracting

1) in Tissue Culture Dish, cell sample is washed after twice with PBS; PBS is blotted only with 1ml rifle, add 1ml Trizol (invitrogen) solution, piping and druming mixes; and be drawn in 1.5ml RNase free EP pipe and make the abundant cracking of cell, room temperature leaves standstill 5min; Tissue sample fully grinds with liquid nitrogen, adds 1ml Trizol (Invitrogen) solution, mixes, and room temperature is placed 5min and made its abundant cracking;

2) add 200 μ l chloroforms, thermal agitation mixes 30s, and water is fully contacted with organic phase, and room temperature leaves standstill 3-5min; (centrifuge tube discharges in order when centrifugal, and centrifugal complete, also sequence of the order of centrifuge tube, with the sequence consensus of the first step)

3) at 4 DEG C, the centrifugal 15min of 14,000g, is divided into three layers as seen, and RNA, at upper strata water, moves to another new RNase free EP pipe;

4) precipitated rna: add equal-volume Virahol, gently fully mix (putting upside down 6-8 time), room temperature leaves standstill 10min;

5), at 4 DEG C, the centrifugal 10min of 14,000g, collects RNA precipitation, removes supernatant;

6) with twice of 75% washing with alcohol (the centrifugal 5min of 12,000g, super clean bench is air-dry;

7) add appropriate DEPC water (at least 15ul) dissolution precipitation depending on precipitation capacity.

Two) go the operation of genome step:

1) add isopyknic phenol/chloroform, turn upside down and mix, room temperature is placed 5min, rear 14,000rpm, and centrifugal 15min, gets supernatant.

2) add isopyknic chloroform, turn upside down and mix, after stratification 14,000rpm, centrifugal 15min, gets supernatant.

3) add equal-volume Virahol, gently fully mix (putting upside down 6-8 time) ,-20 DEG C of standing 15min;

4), at 4 DEG C, the centrifugal 15min of 14,000g, collects RNA precipitation, removes supernatant;

5) by twice of 75% washing with alcohol (the centrifugal 5min of 12,000g), super clean bench is air-dry;

6) add appropriate DEPC water (at least 15ul) dissolution precipitation.

Three) total RNA purity and integrity detection

1) purity detecting: get 50 times of dilutions of 1 μ l RNA sample, measure OD value on nucleic acid-protein detector, the ratio of OD260/OD280 is greater than 1.8, illustrates that the RNA of preparation is purer, without protein contamination.

2) total RNA integrity detection: get RNA sample 1 μ l, 1% agarose gel electrophoresis 80V × 20min, the EB 10min that dyes, observes and 5s rRNA, 18s rRNA and the 28s rRNA band of total RNA that takes pictures with gel imaging system.

Four) mRNA reverse transcription operation steps:

1), in the PCR of RNase free pipe, add Total RNA 1.0 μ g and H2O configuration cumulative volume 12 μ l solution.

2) by above-mentioned solution piping and druming evenly, put 85 DEG C of insulation 5min, make RNA sex change.Immediately refrigeration on ice, to prevent RNA renaturation;

3) in this PCR pipe, add Promega reagent

4) by 30 DEG C of insulation 10min of above-mentioned 20 μ l reaction soln;

5) 42 DEG C of insulation 50min;

6) 85 DEG C of insulation 10min;

7)-20 DEG C of preservations.

Five) quantitative PCR detection

1, primer test:

Test its specificity and amplification efficiency according to needing to carry out qPCR before the formal experiment of the primer of mRNA design, concrete reaction system and reaction conditions are as formal experiment, and every pair of primer need do the contrast of template water.。

2, system preparation:

H2O 4ul

SYBR Green PCR Master Mix 10ul (TOYOBO) (needing vibration before use evenly)

Upstream primer 0.5ul (10uM)

Downstream primer 0.5ul (10uM)

Cumulative volume 15ul

After total system prepares, in vibrator, vibration evenly or with rifle is inhaled and is beaten evenly, and then the every pipe of 15ul divides and is filled in 8 connecting legs.

3, cDNA dilutes suitable concentration with sterilizing pure water, is generally 1:20 dilution, after cDNA sequences in certain sequence, can add in the reaction system just having prepared.Application of sample is complete, builds eight connecting leg lids, and goes up the order of the good 1-12 of mark on the limit on edge most at eight connecting leg lids.

4, each row's eight connecting legs are placed on to the centrifugal several seconds on palm whizzer.

5, open specimen holder, put into eight connecting legs, shut specimen holder, and on software, select the position, hole of having put reaction tubes, reject the position, hole of reactionless pipe.

6, the title of the sample title of the good each reacting hole of mark and detection gene on 7500 softwares, destination file is kept in classification.

After having reacted, eight connecting legs should install in sealed bag, the good filename of mark on sack, client's name.(same client repeats experiment for three times, only needs to preserve the reaction tubes wherein once repeating, and all the other are discardable)

three, mtt assay experimental procedure

1, inoculating cell: with DMEM nutrient solution (Hyclone) 5%CO2 containing 10% tire calf serum (Thermo), 37 DEG C of cultivations of going down to posterity, Panc-1 derives from ATCC, SETD4 ShRNA(TL301750) be OriGene product, be made into individual cells suspension, be inoculated into 96 orifice plates with every hole 1000-10000 cell, every pore volume 200ul.;

2, culturing cell: with general culture condition, 5%CO2,37 DEG C, at the bottom of being paved with hole to cell monolayer (96 hole flat underside).

3, colour generation: cultivate after 3-5 days, every hole adds MTT(tetrazolium bromide) solution (5mg/ml prepares with PBS, pH=7.4) 20ul. continues to hatch 4 h, stops cultivating, careful suction abandoned culture supernatant hole in, for suspension cell need centrifugal after again suction abandon culture supernatant in hole.Every hole adds 150ul DMSO, and vibration 10min, fully melts crystallisate.

4, colorimetric: select 490nm wavelength, measure each hole absorbance value on enzyme linked immunological monitor, record result.

four, the foundation of carcinoma of the pancreas transplanted tumor animal model and curative effect testing method

1. determine human body knot cancerous tissue and obtain cancerous tissue by operation, or collecting cultured cells strain, as Panc-1, SETD4 ShRNA (TL301750) is OriGene product.

2. shorten tumor tissues or the cell residence time in vitro to keep to greatest extent the freshness of tumor tissues as far as possible.

3. carry out after skin degerming with 70% alcohol, to both sides, every sidesway is planted 3 tumor tissues and is carried out disinfection, or direct injection pancreatic tumor cell.Immunodeficient mouse can be BALB/C or NOD/SCID mouse.

4. each group is got 6.In the time that growing to average 150mm3, the transplantation tumor of every Immune deficient mice starts administration.Dosage regimen comprises negative control and different treatment plan and concentration.

5. after administration, the tumor size of every Immune deficient mice changes needs within every 3-5 days, measure once.

6. effect of drugs calculation formula: T/C(treatment/contrast) X100%, in order to assess the curative effect of all schemes.

five, carcinoma of the pancreas stem cell sorting method

1. get human pancreas cancer sample, or obtain tumor specimen from Nude Mouse Model, utilize Collagenase to cut digestion method and prepare cell suspension, 37 DEG C of digestion 2 ~ 3 h of tumor tissues, make single cell suspension with 40uM strainer filtering after digestion.

2. adjusting cell concn with 2%FCS RPMI1640/HBSS is 100 ten thousand～500 ten thousand/ml.Antibody CD44 and c-Met(BD for cell) mark (4 DEG C of 20 minutes binding times.) with washings washing 2 times, add washings 4ml left and right, centrifugal 1000rpm × 5 minute at every turn.

3.H2-Kd removes non-pancreatic cancer cell (nude mice cell), and 4,6-diamidino-2-phenylindone (DAPI) is removed dead cell

4. flow cytometer (BD Aria) divides the primary carcinoma of the pancreas CD44+c-Met+ cell of choosing, and cell sorting 2 times ensures sorting cells purity >90%.

six, statistical analysis method

Data presentation is mean value ± SE.Statistically-significant difference is used Student t inspection and X 2 analysis to determine under different situations, and is defined as P<0.05.

embodiment 1 detects SETD4 at pancreatic tumor cell, expression level in pancreas diagnosis of hepatic metastases cell and tumor stem cell

Clinical samples inclusion criteria: accept surgical resection case, Pathological diagnoses is ductal adenocarcinoma of pancreas; Percutaneous biopsy obtains complete histological specimen, and pathological diagnosis is ductal adenocarcinoma of pancreas.Participate in all acceptance, radiotherapy and immunotherapies before the corrective surgery of this research or biopsy.

Method: obtain postoperative and biopsy normal pancreatic tissue, pancreatitis, pancreatic tumor tissue, and by airflow classification Isolation of pancreatic tumor stem cell.After extracting RNA, carry out qRT-PCR, qRT-PCR operation steps as above.In qRT-PCR contrast normal pancreatic tissue, pancreatitis, pancreatic tumor tissue and pancreatic neoplasm stem cell, the expression level of SETD4 changes.

Result: as shown in Figure 1, quantitative PCR data find that the expression level of SETD4 is lower in normal pancreatic tissue, phase normal tissue, pancreatitis SETD4 only has slight rising, and in tumor tissues raises 6 times, raise nearly 10 times pancreatic neoplasm stem cell.The routine sample of the every group analysis 10 of quantitative PCR.Immunohistochemical methods inspection finds that Normal Pancreas patient only has 3.7% for the SETD4 positive, and pancreatitis is 6.5%, and pancreatic tumor tissue is 72%, and pancreatic neoplasm hepatic metastases is organized as 85%(table 1).Immunohistochemical methods checks that every group is 16 routine samples.

Conclusion: SETD4 quantitative PCR and immunohistochemical methods inspection can be used to diagnosis of pancreatic cancer.

embodiment 2 detects the relation of SETD4 in the positive level of pancreatic tumor cell and prognosis

Method: select to have the complete record 21 routine Patients with Pancreatic Cancers of following up a case by regular visits to, obtain pathological section and carry out the inspection of SETD4 immunohistochemical methods, immunohistochemical methods operation steps as above.Selected patient all postoperative carry out conventional gemcitabine chemotherapy, without other treatment record.

The SETD4 immunohistochemical methods inspection of 21 routine Patients with Pancreatic Cancers is divided into three groups (strong positive is 3, and the positive is 2, and the weak positive is 1), and every group of 7 examples, carry out prognosis tracking, follows the tracks of patient's survival time.

Result: as shown in Figure 2, prognosis track data shows that the positive level of pancreatic tumor cell and prognosis are inverse relation lifetime, strong positive patient poor prognosis, lifetime is short, and weak positive patient prognosis is better, lifetime is longer.

Conclusion, SETD4 immunohistochemical methods inspection AQL can be used to predict prognosis, and can be used as the index of predicted treatment effect.

embodiment 3 detects the growth relationship of blocking-up SETD4 expression and pancreatic tumor cell

1) blocking-up SETD4 can suppress pancreatic tumor cell survival

Method: experiment in vitro utilizes Panc-1 cell strain to carry out MTT experiment, analyzes the impact of blocking-up SETD4 on pancreatic cancer cell survival rate.Gemcitabine in contrast.

Result: as shown in Figure 3, gemcitabine is tested pancreatic cancer cell survival rate little in vitro, and experiment in vitro blocking-up inhibition SETD4 can obviously reduce pancreatic cancer cell survival rate.

Conclusion: block the survival rate that SETD4 can effectively suppress pancreatic tumor cell in experiment in vitro.

2) experimentation on animals proves that blocking-up SETD4 can suppress pancreatic neoplasm growth.

Method: pancreatic cancer cell and the control group pancreatic cancer cell of transfection SETD4 ShRNA are inoculated into immune deficiency

Mouse is set up people source carcinoma of the pancreas animal model and studies, the impact of inspection blocking-up SETD4 on pancreatic cancer growth in animal model.The drug withdrawal after 4 weeks of gemcitabine (twice weekly of 100 mg/kg) successive administration, gross tumor volume is measured 8 weeks.

Result: as shown in Figure 4, blocking-up SETD4 can suppress pancreatic neoplasm growth in animal model.With general

Logical Panc-1 cell is compared, and the tumor growth that after Panc-1 cell transfecting SETD4 ShRNA, subcutaneous injection nude mice forms is suppressed.And blocking-up SETD4 associating carcinoma of the pancreas choice drug gemcitabine (Gemcitabine, Gem) result for the treatment of is better.The experimental result of utilizing primary people source carcinoma of the pancreas animal model (Patient-Derived Xenograft-PDX) is with to utilize the experimental result of Panc-1 consistent.

Conclusion: blocking-up SETD4 can obviously suppress the pancreatic cancer cell growth in nude mouse.

3) blocking-up SETD4 can suppress the EMT of pancreatic neoplasm stem cell and tumour.

Method: the tumor tissues that above-mentioned experimentation on animals is obtained produces unicellular through collagen protein enzymic digestion, utilizes CD44 to separate with C-met antibodies streaming and obtains CD44+c-Met+ carcinoma of the pancreas stem cell, the impact of research blocking-up SETD4 on tumor stem cell.Carry out the impact of quantitative PCR analysis research blocking-up SETD4 on EMT path from extracting RNA in cell.

Result: utilize SETD4 ShRNA blocking-up SETD4 can reduce significantly the quantity of pancreatic neoplasm stem cell, and block SETD4 associating gemcitabine (Gemcitabine, Gem) result for the treatment of better (table two).As shown in Figure 5, utilize the tumour cell of digestion to carry out quantitative PCR detection, find that blocking-up SETD4 can suppress the EMT of Snail mediation.

conclusion: blocking-up SETD4 can reduce pancreatic neoplasm stem cell population significantly, and suppress the EMT of Snail mediation.

In sum, studies confirm that SETD4 high expression level in pancreatic tumor cell and tumor stem cell, SETD4 can be applicable to diagnosis of pancreatic cancer, and as the index prediction prognosis of predicted treatment effect; Target SETD4 can suppress pancreatic neoplasm growth, suppresses pancreatic neoplasm stem cell, can be used as the novel targets for the treatment of of pancreatic cancer new drug and biological immune treatment.

Claims

The purposes of 1.SETD4 in preparation diagnosis of pancreatic cancer and/or prognosis kit.
The purposes of 2.SETD4 blocker in preparation treatment carcinoma of the pancreas medicine.