CN109999034A - Oncoprotein TNIK kinases targets natural small molecule inhibitor and its application - Google Patents

Abstract

The invention belongs to biological field, it is related to a kind of oncoprotein TNIK kinases targeting natural small molecule inhibitor and its application.The invention discloses a kind of oncoprotein TNIK kinases to target natural inhibitor, is jateorrhizine.The present invention further simultaneously discloses above-mentioned oncoprotein TNIK kinases targeting natural inhibitor and is preparing the application in anti-breast cancer medicines.The present invention parses the molecular mechanism of TNIK regulation Metastasis in Breast Cancer, seeks the targeted drug for inhibiting the transfer of mammary gland cancerous invasion, has great importance for the treatment of breast cancer.

Description

Oncoprotein TNIK kinases targets natural small molecule inhibitor and its application

Technical field

The invention belongs to biological fields, and in particular to a kind of oncoprotein TNIK kinases targeting natural small molecule inhibitor and It is applied.

Background technique

Breast cancer (breast cancer) is to seriously endanger the first big malignant tumour of women's health in the world, postoperative turn Shifting is its more refractory the main reason for being cured.National Cancer research center latest result is shown within 2016, breast cancer incidence Malignant tumour 29% is accounted for, and morbidity crowd tends to become younger.Breast cancer is that canceration occurs for galactophore epithelial cell, the distal end occurred Transfer is, pathogenesis and oncogene activation, suppression the same with other malignant tumours the main reason for causing clinical treatment to fail Oncogene inactivation is closely related, recent studies have shown that, TNF receptor associated factor (TRAF) -2 (Traf-2) and Nck interaction swash Enzyme (TNIK) altimeter in a variety of entity tumors such as colon cancer, cancer of pancreas, non-small cell lung cancer, prostate cancer and breast cancer It reaches, and is closely related with clinical stages and poor prognosis,

TNF receptor associated factor (TRAF) -2 and the serineprotein kinase (TNIK) of Nck interaction are a kind of multi-effects Tumour target protein, abnormal height is expressed in breast cancer tissue, and TNIK may participate in activation Wnt/ β-catenin access, for The invasion transfer of breast cancer is most important.

Jateorrhizine (Jatrorrhizine) is from the Ranunculaceae coptis, Berberidaceae plant leatherleaf mahonia and menispermaceous plants black ox The isoquinolin woods Alkaloid extracted in the plants such as gallbladder, that is, jateorrhizine belongs to protoberberine type isoquinoline alkaloid.

Summary of the invention

The technical problem to be solved in the present invention is to provide a kind of oncoprotein TNIK kinases to target natural small molecule inhibitor And its application.

In order to solve the above technical problem, the present invention provides a kind of oncoprotein TNIK kinases to target natural inhibitor, For jateorrhizine.

The present invention, which goes back while providing above-mentioned oncoprotein TNIK kinases targeting natural inhibitor, is preparing anti-breast cancer medicine Application in object.

The present invention sets jateorrhizine as TNIK kinases targeting inhibitor, jateorrhizine to the growing multiplication of breast cancer cell with And transfer all have significant inhibiting effect, cell cycle regulation, induced apoptosis, and jateorrhizine can target it is swollen Tumor albumen TNIK regulates and controls the expression of Wnt/ β-catenin pathway associated protein, to inhibit to inhibit breast cancer cell growth proliferation, makees For a kind of natural targeted inhibition agent of oncoprotein TNIK, the targeted therapy of breast cancer can be applied to.

TNIK promotes tumour growth, is pharmaceutically-active important target spot.

TNIK (TRAF-2and Nck interacting kinase) is by the silk of adaptor protein TRAF-2 and Nck interaction Propylhomoserin protein kinase, TNIK gene are located at the area 3q26 of chromosome, and gene magnification can all occur for the region in kinds cancer. TNIK coding contains 1360 amino acid polypeptide chains, and structure is swashed by N-terminal kinase domain, central domain and C-terminal centrum germinativum Enzyme homeodomain forms (Fig. 1).Cal gene research institute is newest studies have shown that 80% causes the gene of human diseases one Kind is named all to be existed in " Xenopus tropicalis " African toad gene, and by comparing mankind TNIK and Africa xenopus TNIK Amino acid sequence discovery both similarity be up to 91%.Germany scientist (2012) report, the EBV for carrying TNIK can be activated The generation of immune system induction cancer and lymthoma.Medical University Of Tianjin finds that TNIK expression is significantly high in Pancreatic Adenocarcinoma In cancer beside organism, be closely related with clinical pathology environment and survival region, and TNIK high expression can promote Cell Proliferation of Pancreatic Cancer Cell, The biological activities such as invasion, migration and inhibition apoptosis.Yu etc. is confirmed in Cancer in China patient using fluorescence in situ hybridization technique There is the high expression of TNIK gene, and proves that TNIK is a potential innovation target for cancer therapy.Therefore, TNIK promotes tumour raw It is long, it is the important target spot of tumor therapeutic agent effect as tumor targets albumen.

Wnt gene is most early in a kind of proto-oncogene cloned in mouse breast cancer.The Wnt signal of report activation at present is logical The complicated multiplicity of road mechanism, classical Wnt/β-catenin access are that β-catenin level intracellular increases and enters that core is interior and TCF In conjunction with adjusting the transcript and expression of downstream target gene, influence cancer cell and the biological characteristics such as stick, be proliferated, migrate and invade (such as Shown in Fig. 2).It is reported that TNIK activates Wnt access to play an important role the growth for promoting colorectal cancer.The report such as Yamada Since apc gene is mutated, lead to Wnt signal path APC and β-catenin, axin, the dissociation of GSK3 β complex, β-catenin It is accumulated in endochylema, activating transcription factor TCF4 plays carcinogenicity, and TNIK is the T cell factor (TCF-4) and β-in nucleus The activating enzymes of catenin transcription complex.Mahmoudi etc. utilizes proteomics analytical technique of mass spectrum, thin in mouse small intestine epithelium Born of the same parents identify the TNIK with TCF-4 interaction.TCF-4 transcriptional level plays a significant role in activation Wnt expression of target gene in core, TNIK is directly combined with β-catenin by central domain, through N-terminal kinase domain in conjunction with TCF4.The hair such as Shitashige Existing, by TNIK phosphorylation, β-catenin is combined No. 154 serine residues in colon cancer cell in TCF4 polypeptide chain in TNIK It serves as a connection during activation TCF4.TNIK can also be by regulation Wnt co-receptor GAP-associated protein GAP 6 (LRP6) in the steady of cell membrane It is qualitative, to influence the transmitting of Wnt signal path.The present invention has found Wnt target gene to target TNIK tiny RNA interference colon cancer cell Encoding albumen c-MYC and cyclinD1 expression significantly reduces；And apafl caspase-9 and PARP-1 expression increase Height shows that TNIK can be by inhibiting apoptosis to promote tumour growth.

The novel targets that TNIK is acted on as tumour medicine, foreign countries are about chemically synthesized TNIK little molecules in inhibiting at present The report of agent.National Cancer Center Research Institute's high flux screening goes out inhibitor for treating of the aminothiazole analog derivative as TNIK It is special to obtain the U.S. to the solid carcinomas significant effect such as colon cancer, cancer of pancreas, prostate cancer, breast cancer for cancer patient, clinical discovery Benefit.Astex drugmaker, the U.S. as global cancer drug research and development leader has developed based on phenylamino pyridine structure TNIK chemical inhibitor.Yu etc. can significantly inhibit cancer cell multiplication and inducing cell death using RNA interference TNIK expression.Cause This, TNIK inhibitor is the treatment ideal targeted drug of entity tumor, but the country is current and has no the report in relation to TNIK inhibitor Road.The present invention using Molecular Simulation Technique tentatively jateorrhizine is docked with TNIK protein molecular (PDB 2X7F) on the basis of into One step research finds that jateorrhizine has similar chemical structure active group with TNIK first wife's body by molecular simulation, can be with TNIK Amino acid in structure forms multiple connections, and jateorrhizine molecule can enter the activated centre of TNIK albumen, has targeting well It docks effect (Fig. 3), shows that jateorrhizine may show TNIK inhibitor activity, but jateorrhizine is anti-as TNIK targeted inhibition agent The mechanism of mammary gland cancerous invasion transfer, it is still necessary to which antitumor pharmacology activity experiment is further verified.

It is of the invention research shows that TNIK is expressed in a variety of cancer cells promotes tumour growth, TNIK can be used as treatment of cancer A new target spot.

Jateorrhizine belongs to protoberberine type isoquinoline alkaloid, which all has a variety of cancer cells stronger Cytotoxic effect.Simulating docking display TNIK by drug molecule may be the target inhibition egg that jateorrhizine acts on cancer cell It is white.Therefore the molecular mechanism of parsing TNIK regulation Metastasis in Breast Cancer, seeks the targeted drug for inhibiting the transfer of mammary gland cancerous invasion, for The treatment of breast cancer has great importance.

Detailed description of the invention

Specific embodiments of the present invention will be described in further detail with reference to the accompanying drawing.

Fig. 1 is the molecular structure of TNIK；

Fig. 2 is the molecular mechanism figure of TNIK abnormal activation Wnt/ β-catenin signal path；

Fig. 3 is the molecular docking illustraton of model of jateorrhizine and TNIK crystal structure；

Fig. 4 is the TNIK gene map in CRISPR/Cas9 technology knockout breast cancer cell MDA-MB-231；

Fig. 5 is MDA-MB-231/TNIK-Control and the intracellular TNIK of MDA-MB-231/TNIK-KO, p-TNIK and The expression of Wnt/ β-catenin signal path and jateorrhizine handle the influence to it；

(A) Immunofluorescence test TNIK, p-TNIK and Wnt/ β-catenin signal path correlative protein expression；

(B) Western blotting analysis detection TNIK, p-TNIK and Wnt/ β-catenin signal path GAP-associated protein GAP Expression；

Fig. 6 is that jateorrhizine processing MDA-MB-231/TNIK-Control and MDA-MB-231/TNIK-KO cell lives to it The influence of property and period；

(A) cytotoxic effect of violet staining experiment detection jateorrhizine；

(B) flow cytometer detection jateorrhizine induced apoptosis；

(C) WB detects jateorrhizine induced apoptosis.

Specific embodiment

The present invention is described further combined with specific embodiments below, but protection scope of the present invention is not limited in This:

In the present invention, first with gene editing CRISPR/Cas9 technology targeting knockout TNIK, stable transfection is established Cancer cell line MDA-MB-231/TNIK-KO and MDA-MB-231/TNIK-Control are examined by immunofluorescence and immunoblotting TNIK, p-TNIK and Wnt/ β-catenin access key protein expression in cancer cell after survey TNIK is knocked out, to prove to strike Except the controllable Wnt/ β-catenin access key protein expression of TNIK and epithelial-mesenchymal conversion (EMT) is reversed, influences breast cancer The transfer invasive ability of cell, and its effect can be reinforced by the way that jateorrhizine processing is added.MTT, flow cytometer, immunofluorescence and For immunoblotting analysis the result shows that jateorrhizine can significantly inhibit the cell activity of breast cancer cell MDA-MB-231 and MCF-7, regulation is thin In the intracellular growth period, inducing cell mitochondrial membrane potential changes, so that early apoptosis occur.It is cured by Clone formation, scratch Conjunction, immunofluorescence and immunoblotting analysis etc. are experiments have shown that jateorrhizine can destroy cell membrane skeleton protein F-actin, regulation TNIK egg White expression destroys the integrality of breast cancer cell, to reduce the movement transfer ability of cancer cell.For Wnt signal path, medicine The expression that root alkali can regulate and control key protein β-catenin makes its downward, up-regulated expression GSK-3 β albumen, and regulates and controls EMT epithelium Phenotype E-cadherin protein expression dramatically increases, and interstitial phenotypic marker N-cadherin protein expression is in decreasing trend, To reverse EMT.Jateorrhizine can obviously inhibit the growth and transfer of breast cancer cell, and to tumor-bearing mice without overt toxicity.

It is specific as follows:

Cell used is described in table 1 below.

Table 1

Experimental method is as follows

1, the recovery of cell

Take out freeze-stored cell, be put into rapidly in 37 DEG C of water-baths be incubated for after, be put into centrifuge centrifugation 1000rpm, three minutes After take out, discard frozen stock solution on the super-clean bench, cell be resuspended with corresponding culture solution and is transferred to added with the fresh training liquid of 10%FBS 100cm²It in dish, is put into cell incubator and cultivates after cell dispersion is shaken up, to cell culture fluid flavescence or cell density Liquid is changed when reaching 80%.

2, the passage of cell

Training liquid is discarded, cleans cell with 3 milliliters of PBS, 3 milliliters of 0.25%Trypsin-EDTA are then added, are put into cell It is sufficiently digested in incubator, observes cellular morphology, contraction is put into workbench after being rounded, sucks digestive juice, and it is thin to pat the dispersion of ware wall Born of the same parents, and be resuspended cell with corresponding culture solution, fully dispersed and be transferred in centrifuge tube, 800rpm is centrifuged 3 minutes, discards training liquid, Fresh culture is added, cell dispersion is resuspended, then carry out cell passage according to suitable ratio, cell dispersion is put into after shaking up In cell incubator, liquid is changed when cell culture fluid turns yellow or cell density reaches 80%.

3, cell freezes

Frozen stock solution is prepared in advance, and according to serum: the ratio of DMSO=9:l is put into 4 DEG C of spare, old cultures of suction abandoning after mixing Liquid adds 3 milliliters of PBS to wash twice, and trypsin digestion to cellular contraction, gap becomes larger, and adds fresh medium termination and disappears Change, blow and beat cell, and be transferred in 15 milliliters of centrifuge tubes, 1000rpm is centrifuged three minutes, discards supernatant liquid, and frozen stock solution is added, blows It beats and mixes, take 10 microlitres of countings, adjust cell number in l~5 × 10⁶/ mL takes l milliliters of cell suspensions into cryopreservation tube, is put into jelly The cooling of box inside gradient is deposited, is finally putting into liquid nitrogen container.

4, MTT is detected

With the concentration kind in about 10,000 every holes, into 96 orifice plates, after 12 hours are adherent, gradient concentration dilution is added in cell When being put into cell incubator culture after good drug or training liquid to specified time point, 20 μ L MTT working solutions of every hole addition, It is incubated for 4 hours in cell incubator, then sucks supernatant and 150 μ L DMSO are added, be put in horizontal oscillations 20 minutes on shaking table After banyan solution crystal, the absorbance value under 490nm wavelength is detected with microplate reader, cell survival rate is calculated according to formula.

Cell survival rate=(experimental group OD value-control group OD value)/(control group OD value-zeroing hole OD value) × 100%.

5, crystal violet staining assay detects cytotoxicity

Cell dissociation is resuspended as after individual cells, adjustment concentration is entered in 24 orifice plates with 20,000, every hole cell kind, and 12 is small When cell it is substantially adherent after, relative medicine concentration gradient is added, after drug treating time point reaches, discards training liquid, every hole is added 500 μ L violet staining liquid, are placed at room temperature for two and discard crystal violet ten minutes later, gently wash away remaining crystal violet with distillation and contaminate Liquid is taken pictures after being put into 37 DEG C of baking oven drying.

6, the cell cycle is detected

Cell is entered in six orifice plates with the quantity kind in 30,000 every holes, through corresponding gradient concentration drug-treated to corresponding time Afterwards, cell culture fluid is drawn into pipe in advance, with 0.25%Trypsin (being free of EDTA) digestion incubated cell, is sucked 0.25%Trypsin, it is then that cell is fully dispersed, it draws into preparatory culture solution, sufficiently dispels.Cell is put into centrifugation Machine, 1000g or so are centrifuged 3-5 minutes, and by extra broth out, there are cell masses, and l milliliters of pre-cooling PBS are added, and cell is resuspended. It is placed again into centrifuge and collects precipitating, i.e. cell mass, suck extra PBS, there are cells, and tube bottom is resuspended with finger tip, protects cell Dispersity is held, is avoided agglomerating.

Cell is fixed: l mL ice bath is added and is pre-chilled in 70% ethyl alcohol, gently piping and druming mixes, and 4 DEG C are fixed 2 hours or so, with 1000g or so is centrifuged 3-5 minutes sedimentation cells afterwards, carefully absorbs supernatant, 70% second of about 50 μ L of residual or so is liquor-saturated, to avoid suction Walk cell.Cell is resuspended in the PBS that the pre-cooling of about l mL ice bath is added, again centrifugation cell, careful to absorb upper clean, residual about 50 The PBS of μ L or so, to avoid cell is siphoned away, gently attack centrifuge tube bottom avoids cell agglomerating with appropriate cell dispersion.

Dyeing liquor is prepared by 0.5mL dye solution, 25 μ L propidium iodide stain liquid (20X), 10 μ L RNase A (SOX) It forms.

Dyeing: drawing 0.5 milliliter of PI dyeing liquor and be added in cell liquid, be put on turbula shaker and be slightly vortexed, and makes thin Born of the same parents are completely dispersed, and are put into cell incubator and are protected from light incubation 30 minutes, keep cell to be placed in ice chest when sample presentation detects, utilize streaming Cell instrument carries out cell cycle detection.

7, line grain stops film potential (CMMP) detection

After cell dissociation is resuspended, every hole kind enters 2 × 10 in 6 orifice plates⁵Gradient concentration is added after cell is adherent in a cell After the drug-treated corresponding time, culture solution is absorbed, it is primary wash cell with PBS, and then addition l mL cell culture fluid, adds L mL JC-I dyeing working fluid mixes well, and is incubated for 20 minutes for 37 DEG C in cell incubator.In incubation period, according to every 1mL The ratio of 4mL distilled water is added in JC-1 dye solution (5 ×), prepares suitable JC-1 dye solution (1 ×), and be placed in Ice bath.It asks on being absorbed after being incubated for 37 DEG C, is washed 2 times with JC-l dye solution (1 ×), 2mL cell culture fluid is added, It is observed under laser confocal microscope.

8, Apoptosis detects

After collecting the cell after gradient concentration drug-treated, cleaned twice with pre-cooling PBS, with l × Binding Cell is resuspended in Buffer, and whole density is 1 × 10⁶A/mL.Take 100 μ L that cell liquid (l × 10 are resuspended⁵It is a) into new centrifuge tube, 5 μ L FITC Annexin V and 5 μ L PI are separately added into, after incubation at room temperature being protected from light after vortex 15 minutes, every pipe is added 400 μ L's L × Binding Buffer, is then detected with flow cytometer.

For the GFP albumen pair for eliminating the expression of MDA-MB-231/TNIK-control and MDA-MB-231/TNIK-KO cell The influence of fluorescence detection replaces apoptosis the Testing index FITC and PI of regular growth, the same BD of testing process using PE and 7-AAD Company's FITC Annex in V apoptosis detection kit.

9, colony formation

After cell dissociation is resuspended, every hole kind enters 2 × l0 in 6 orifice plates⁵A cell discards cell after drug-treated Survivaling cell is digested and is resuspended, entered in new 6 orifice plates with 1000, every hole cell concentration kind, in cell incubator by culture solution In continue culture two weeks, changed liquid every three to four days.Culture solution then is discarded, is washed twice with PBS, is waken up with 4% poly first Violet staining liquid is added after fixed cell to dye 15 minutes, then is washed three times with PBS, is dried in 37 DEG C of biochemical cultivation cases It is dry, it is observed under inverted microscope, the clone more than 50 cells is counted.

10, scratch Healing Experiments

The cell for choosing logarithmic growth phase, is laid in 12 orifice plates with 5000, every hole cell density, is put into cell incubator Interior culture, after cell is adherent cover with after, the cell culture fluid containing 10%FBS is changed to serum-free medium, and medicine root is added Alkali (10 μM), and jateorrhizine processing is not added as a control group, three traces are drawn with the pipette tips of 10 μ L in every hole, continue to place In being cultivated in cell incubator, observed when 0 hour and 48 hours by inverted microscope between every hole inner cell trace away from From, and record result.

11, immunofluorescence (IF) is tested

1) slide, is put into the every hole of 24 orifice plates, adjustment concentration is resuspended to every 1 × l0 of hole in cell dissociation⁵A cell It is added in orifice plate, is put in after standing 20min on super-clean bench and places into cell incubator culture.

2), after cell is adherent, culture solution is sucked, is slowly added to l × PBS along wooden partition, every hole l mL is washed 2 times, then along plate Wall is slowly added to 4% paraformaldehyde, and every hole lmL is placed at room temperature for 15 minutes or so fixed cells.

3) paraformaldehyde softly then, is sucked, l × PBS is softly added along wooden partition, every hole l rnL is placed at room temperature for 5min, PBS is sucked, is washed 3 times.

4), 0.5mL 0.5%Triton X-100 is added in every hole, in being incubated for 30 minutes in cell incubator, increases antibody Permeability.

5), 0.5mL 5%BSA closing is added in every hole, and in being incubated for 30 minutes in cell incubator, then addition has diluted Primary antibody, 4 DEG C of refrigerators are incubated overnight.

6) primary antibody, is washed away, l mL PBS 10min/ times, 3 times, is washed in vibrating on shaking table.

7) secondary antibody, is diluted, 200 μ L are added in every hole, and 37 DEG C are protected from light incubation 1h.

8) secondary antibody, is washed away, 1mL PBS 10min/ times, 3 times, is washed in vibrating on shaking table.

9), every hole adds 500 μ L DAPI to dye, 10min.

10), every hole adds 1 × PBS to wash 2 times, 10min/ times, does not shake on shaking table.

11) the floating agent of going out of anti-fluorescence, is instilled, with nail sheet for oil seal.

For the GFP albumen pair for eliminating the expression of MDA-MB-23l/TNIK-control and MDA-MB-231/TNIK-KO cell The influence of fluorescence detection is first quenched GFP with 100% methyl alcohol process cell of pre-cooling, then again before carrying out fluorescent staining experiment Cell is carried out to fix.

12, it is horizontal to detect correlative protein expression by Western blot

1), extract protein sample: removal culture solution, PBS are resuspended cell, are blotted with paper, and 100 microlitres are added in the every hole of six orifice plates RIPA lysate (l milliliters of RIPA+10 microlitres of PMSF) blows and beats uniform fold, and in cracking or so half an hour on ice, PBS weight is added Outstanding cell is gone in EP pipe, is put into the centrifuge of 4 DEG C of pre-coolings, 12000rpm, 15 minutes, 4 DEG C, taking supernatant was sample egg It is white.

2), protein quantification: standard curve is drawn according to BCA protein quantification specification, 1 microlitre of sample is added in 96 orifice plates Albumen and 19 microlitres of PBS, adding 200 μ L BCA working solutions, (ready-to-use, A, B fluid exchange pipette tips are drawn, and add Kong Shihuan pipette tips Suck), it is put into 60 DEG C of baking ovens and is incubated for 30min, microplate reader measures the absorption values of 570nm wavelength, quasi- by CurveExpert The standard protein curve closed out calculates corresponding protein concentration and loading volume.Remaining supernatant is drawn in EP pipe, records good draw 5 × Loading Buffer of 1/4V is added in volume V, mixes, and being put into 10min in 100 DEG C of metal baths makes albuminous degeneration, cooling After can be placed in -20 DEG C of refrigerators and save.

3), electrophoresis: loading albumen V needed for being calculated by protein quantification, prepare corresponding concentration Protein Separation glue and 5% it is dense Contracting glue draws protein sample and enters hole, and 5 μ L of pre-dyed marker is added, is vortexed and mixes before albumen loading, electrophoresis 70V, 35min, egg White 130V after entering separation gel, 50min or so stop electrophoresis.

4), transferring film: the pre-cooling of transferring film liquid, pvdf membrane is submerged initially in methanol activation, according to white board (+)-sponge-filter paper × 2- Pvdf membrane-gel-filter paper × 2- sponge-black plate (-), after installing sandwich folder, according to black cathode to black plate, white is just Extremely red plate direction is placed, progress ice bath transferring film, 400mA, 120 minutes.

5) it, closes: taking out pvdf membrane, be put into 5%BSA or skimmed milk power confining liquid, room temperature shakes 2 hours left sides of closing It is right.

6), hybridize: being cut off pvdf membrane according to destination protein, be put into corresponding primary antibody and be incubated in box, 4 DEG C of refrigerators were incubated for Night is washed film 3 times, each 10min with 1 × TBST, and shaking table shakes, and pvdf membrane is put into secondary antibody and is incubated in box, and room temperature shaker 2h is used 1 × TBST is washed film 3 times, each 10min, and shaking table shakes.

7), develop: washed pvdf membrane being taken out from incubation box, ECL reagent gently uniform fold pvdf membrane is added, so Developed afterwards using ultra sensitive chemical light-emitting appearance.

13, CRISPR/Cas9 targeting knockout TNIK albumen system

After MDA-MB-231 cell dissociation is resuspended, enter in six orifice plates according to the concentration kind of ten thousand cells of every hole 15-25, it is unparalleled Anti- the culture of training liquid 24 hours, cell fusion degree was up to 60% or so when transfection.Transfection liquid is prepared in advance, and before transfection, by six Training liquid in orifice plate changes fresh training liquid without double antibody into, and transfection liquid rotation is added dropwise and mixes, continues culture cell 24-72 hours, Start not changing training liquid in 24 hours, the replaceable training liquid in 24-72 hours.

Knock out the preparation and transfection process of plasmid transfection liquid:

1), Solution A:1-3 μ g TNIK CRISPR/Cas9KO Plasmid or Control CRISPR/Cas9KO Totally 150 μ L, mixing are stored at room temperature 5min to Plasmid+Plasmid Transfection Medium；

Solution B:5-15μLTransfection Reagent+Plasmid Transfection Medium liquid totally 150 μ L is mixed, the quiet cover 5min of room temperature.

2), Solution A is added dropwise in Solution B, is vortexed immediately, is incubated at room temperature 20min or more.

3) liquid, which is trained, before, transfecting, in 6 orifice plates changes fresh training liquid without double antibody into.

4), 300 μ LReagent (A+B Solution) are added dropwise in hole, and tenderness rotation plate is mixed.

14, animal tumor model is tested

The operation of zoopery meets the nursing and use and animal logic of NIH experimental animal, tests from Shanghai SLK The animal company purchase about big female BAl BIc of surrounding/c mouse, raises in SPF grades of experimental centers of zooscopy.In right side of mice cream Room pad position inoculates 5 × 10⁶A 4Tl cell, when solid tumor grows to 80-120m, fluorescent value reaches about 1 × l0⁵Four groups are randomly divided into when photons/sec, at interval of three days respectively at intraperitoneal injection PBS, 5.0mg/kg b.w. cis-platinum (PDD), 2.5mg/kg b.w. jateorrhizine (JAT-L) and 5.0mg/kg b.w. jateorrhizine (JAT-L).Self administration of medication rises, and every seven It detects the variation of mouse-borne tumor fluorescent value by small animal living body imager.After animal pharmacological experiment, puts to death and take out small Mouse lotus knurl carries out slicing experiment operation in pathology room, by the fixed dehydration of tumor mass and is embedded in paraffin mass, slice dyeing observation.

15 statistical analysis

All experiment contents carry out independent repetition three times and test, and, experimental result data is after analyzing with " average The form of the native standard deviation of value " is presented.Statistical analysis is analyzed by biostatistics software SPSS, when * is less than 0.05, With significant statistical difference.

As a result be analyzed as follows:

1. target positions TNIK albumen and its Effect study in signal path

It can determine through immunofluorescence experiment subcellular localization, TNIK and p-TNIK protein expression are predominantly located at breast cancer cell Cytoplasm in, after the CRISPR/Cas9 technology targeting knockout gene (as shown in Figure 4), in cancer cell cytoplasm can detect To not expressing substantially TNIK albumen (as shown in Figure 5A), and MDA-MB-231/TNIK-KO and MDA-MB-231/TNIK- Control cell again after jateorrhizine is handled, jateorrhizine enhance with can dramatically intracellular Wnt/ β-catenin signal path and The targeting mediating effect+6 of EMT correlative protein expression, influences Wnt/ β -- and the EMT of the expression of catenin signal path and tumour cell makees With the expression for being embodied in the key protein β-catenin of intracellular Wnt signal path is lowered, the table of GSK-3 β albumen Up to up-regulation, and the expression of EMT epithelial phenotype marker E-cadherin albumen is raised, interstitial phenotypic marker N-cadherin egg White expression is then lowered, while the expression of cytoskeletal protein F-actin is substantially reduced, and the form of tumour cell changes, The above results have also obtained further confirmation by WB experiment, show that knocking out TNIK protein expression will affect Wnt/ β- Key protein expression during catenin signal path and EMT, and participate in cytoskeleton and adjust shape of tumor effect.

As shown in figure 5, CRISPR/Cas9TNIK two incision knock out plasmid (TNIK CRISPR/Cas9KO Plasmid) and Control plasmid (Control CRISPR/Cas9Plasmid) is U.S. Santa cruz Biotechnology Products.Benefit With newest gene knockout CRISPR/Cas9 technology, stable breast cancer cell model TNIK is constructed^-/-: by CRISPR/ Cas9TNIK knocks out plasmid transfection into breast cancer cell, before transfection for 24 hours, breast cancer cell is inoculated with 6 orifice plates culture, cell melts It is right that up to 60% or so, transfection reagent carries out cell transfecting, using equivalent CRISPR/Cas9control plasmid as negative control, TNIK is obtained after transfection^+/+.Fluorescence microscopy transfects CRISPR/Cas9control and CRISPR/Cas9TNIK plasmid under the microscope Breast cancer cell growth situation afterwards, the cell after transfection are presented green fluorescence representative and transfect successfully；After transfecting 48h, puromycin Screen the stable cell line that TNIK is knocked out.Situation is knocked out using western blotting technical checking TNIK, screens TNIK^-/- And TNIK^+/+Stablizing breast carcinoma cell strain is the key technology for carrying out follow-up study.

In addition, MDA-MB-231/TNIK-Control and MDA-MB-231/TNIK-KO cell is further located through jateorrhizine After managing for 24 hours, by violet staining the experiment proves that jateorrhizine shows that jateorrhizine can obviously inhibit to the toxic effect of cell Cell growth, and be in concentration dependent.It further can be with quantitative analysis not by the bis- dye methods of streaming PE Annexin V/7-AAD Apoptosis cell when with the processing of concentration jateorrhizine for 24 hours is embodied in various concentration jateorrhizine processing MDA-MB-231/ TNIK-Control cell, bring it about apoptosis cell percentages be respectively 6.70 ± 0.41%, 18.24 ± 1.48% and 22.22 ± 0.63%, and the more significantly apoptosis rate point of various concentration jateorrhizine processing MDA-MB-231/TNIK-KO cell It Wei 5.96 ± 1.09%, 24.69 ± 1.17% and 34.23 ± 0.64%.Jateorrhizine induces cell apoptosis as can be seen from the results In concentration dependant trend, and same concentration jateorrhizine induction for 24 hours under the conditions of, MDA-MB-231/TNIK-KO apoptosis rate compared with MDA-MB-231/TNIK-Control cell is more, illustrate MDA-MB-231/TNIK-KO cell to the sensibility of jateorrhizine compared with MDA-MB-231/TNIK-Control cell is high.Jateorrhizine regulating cell apoptotic signal pathway associated protein is detected by WB Expression of results it is found that jateorrhizine can significant induced apoptosis, participate in Procaspase precursor Procaspase-3, The downward of Procapase-8 and Procaspase-9 albumen, and the apoptosis induction that activates independent of Caspase of induction because The reduction of sub- PARP precursor protein, and concentration dependent (as shown in Figure 6) is presented.

The above result of study shows that TNIK is a kind of target cancer cell proteins, and jateorrhizine is targeting TNIK protein knockout Wnt/ β-catenin signal path in breast cancer cell can be effectively suppressed simultaneously to express, reverse the generation of EMT effect.This research Prove that jateorrhizine is integrated to the active site of TNIK molecule as a kind of natural isoquinoline alkaloid, causes it to lose work for the first time Property, it can be used as natural TNIK inhibitor, provide completely new research direction for targeting anti-breast cancer.

The above list is only a few specific embodiments of the present invention for finally, it should also be noted that.Obviously, this hair Bright to be not limited to above embodiments, acceptable there are many deformations.Those skilled in the art can be from present disclosure All deformations for directly exporting or associating, are considered as protection scope of the present invention.

Claims (2)

1. oncoprotein TNIK kinases targets natural inhibitor, it is characterised in that: be jateorrhizine.

2. oncoprotein TNIK kinases targeting natural inhibitor as described in claim 1 is preparing answering in anti-breast cancer medicines With.