CN109758579A - A kind of lipid metaboli activation lipoprotein receptor is promoting the application in the increase of YAP protein phosphorylation - Google Patents

Abstract

The present invention provides a kind of lipid metaboli activation lipoprotein receptors to promote the application in the increase of YAP protein phosphorylation, belongs to biomedicine technical field.The present invention is experimentally confirmed, lipid metaboli activates lipoprotein receptor physical efficiency that YAP protein phosphorylation is promoted to increase, and then is stranded in YAP albumen in cytoplasm, to inhibit the Hippo signal pathway activated of cell, inhibit the proliferation and tumor formation size of liver cancer cells, plays the effect of prevention and/or treatment liver cancer.

Description

A kind of lipid metaboli activation lipoprotein receptor is in promoting the increase of YAP protein phosphorylation Using

Technical field

The invention belongs to biomedicine technical fields, and in particular to a kind of lipid metaboli activation lipoprotein receptor is promoting YAP Application in protein phosphorylation increase.

Background technique

The reason of liver cancer is the sixth-largest common cancer and the third-largest cancer related mortality (Forner et al.Hepatocellular carcinoma.Lancet,2012,379(9822),1245-1255.).It is early since onset is hidden Phase difficult diagnosis, highly invades and grows rapid feature, and when discovery has lost surgical indication, and therapeutic effect is very poor, advanced stage 5 years survival rates of liver cancer patient are only 18%, and have 5 years survival rates of liver cancer patient of DISTANT METASTASES IN down to 3% (Siegel et al.Cancer statistics,2018.CA Cancer J Clin,2018,68(1),7-30.).Although having in recent years New method treats liver cancer, and clinical treatment liver cancer is still based on operation excision and liver transplant, it has recently been demonstrated that these Treatment method less effective and there are shortcomings, including recurrence, chemotherapy resistance and metastases after treatment.Hepatectomy The main reason for being typically considered recurrence of PHC is not assisted in the treatment of, single operation excision is improving liver cancer clinical treatment Bottleneck stage (Qiu et al.Surface markers ofliver cancer stem cells and is reached in effect innovative targeted-therapy strategies for HCC.Oncol Lett,2018,15(2),2039- 2048.).Therefore the mechanism for trying to explore liver cancer genesis and development researches and develops the targeted drug of specificity, joint multidisciplinary synthesis treatment It is most important for the treatment of liver cancer.

It is a kind of I type that lipid metaboli, which activates lipoprotein receptor (The lipolysis stimulated receptor, LSR), Single pass transmembrane receptor protein is mainly distributed on liver while also in small intestine and many other tissues.LSR can be by changing blood middle reaches Absorption from fatty acid adjusts Post-prandial plasma triglyceride levels.In addition, to be identified as a kind of three cell of epithelial tissue tight by LSR It is close connection (tricellulin) one of main constituents, play barrier cell, selective control small-molecule substance passes through and The effect of cell polarity is maintained, the special shape of (tightjunctions, tTJs) is connected for endothelial cell tight.There is experiment table Bright, LSR understands the proliferation of the promotion breast cancer cell of cell selective, and Tumor Differentiation and tumour can be made by being overexpressed LSR in breast cancer Pathway protein abnormal expression is formed, and increases tumor invasiveness and tumour Forming ability (Reaves et al.The role of lipolysis stimulated lipoprotein receptor in breast cancer and directing breast cancer cell behavior.PLoS One,2014,9(3),e91747.)。

Hippo signal path is a kind of highly conserved signal path of evolving, in animal body by adjusting cell Proliferation Homeostasis is adjusted with apoptosis, the access is abnormal closely related with the occurrence and development of a variety of human tumors, including lung cancer, ovary Cancer, liver, prostate cancer and colorectal cancer, and often outcome is very poor by the patient of Hippo signal path exception (Yimlamai et al.Emerging evidence on the role of the Hippo/YAP pathway in liver physiology and cancer.J Hepatol,2015,63(6),1491-1501.).More and more evidence tables Bright, Hippo signal path is extremely important to the effect of the maintenance of cell stemness, adjusting cell metabolism and the conversion of epithelium mesenchyma.It is right One of the research hotspot of Hippo-YAP signal path is exactly the relationship of itself and liver cancer, and many researchs all show liver cancer cells at present It is middle that there are the dysregulation of Hippo-YAP signal path (Lee et al.The Hippo-Salvador pathway restrains hepatic oval cell proliferation,liver size,and liver tumorigenesis.Proc Natl Acad Sci U S A,2010,107(18),8248-8253；Lu et al.Hippo signaling is a potent in vivo growth and tumor suppressor pathway in the mammalian liver.Proc Natl Acad Sci U S A,2010,107(4),1437-1442.).It is as first It is identified as adjusting the access of organ size, has been confirmed as influencing the principal element of liver growth.Any exception of this access Liver will be made to continue undue growth and eventually lead to hepatocellular carcinoma or cholangiocarcinoma.

YAP albumen is the effector molecule of a key effect of Hippo passage downstream, is promoting cell Proliferation, is inhibiting thin Play important role in born of the same parents' apoptosis, abnormal activation will lead to cell and lose contact inhibition, promote malignant transformation of cells. Hippo access is stranded in YAP albumen in cytoplasm phosphorylation YAP albumen or by way of binding directly and can not play it Effect.YAP albumen mainly regulates and controls Hippo signal path downstream gene in conjunction with transcription factor TEAD family protein.It can inhibit Cell phenotype caused by the small molecule and peptide fragment that YAP-TEAD is combined can weaken because of YAP activation.The expression of YAP albumen increases And its increased activity is related to the development of a variety of liver cancer, such as liver mother cell cancer, hepatocellular carcinoma and cholangiocellular carcinoma (Yimlamai et al.Emerging evidence on the role of the Hippo/YAP pathway in liverphysiology and cancer.J Hepatol,2015,63(6),1491-1501.).Experiment shows that YAP protein activation is that liver cancer occurs early The important symbol of phase and the development for influencing liver cancer.

Liver cancer treatment there is no effective therapeutic scheme outside operation excision and liver transfer operation at present, liver cancer patient average of operation periods treats, Improving patient's shorter survival and prevention recurrence is the hot spot studied at present, finds new therapy target and the medicine for target spot Still there are many blank for object research and development.

Summary of the invention

The problem of in view of background technique, the purpose of the present invention is to provide a kind of new preventions and/or treatment The approach and drug of liver cancer.

The present invention provides a kind of lipid metaboli activation lipoprotein receptors to promote the application in the increase of YAP protein phosphorylation.

Preferably, the application includes being prepared into have by lipid metaboli activation lipoprotein receptor to promote YAP albumen phosphorus Acidification increases the drug of function.

In the present invention, the lipid metaboli activation lipoprotein receptor preferably passes through the site Y623 and the protein bound side of YAP Formula promotes YAP protein phosphorylation to increase.

Alternatively, the lipid metaboli activation lipoprotein receptor is preferably promoted by the site S493 and the protein bound mode of YAP YAP protein phosphorylation increases.

The present invention provides a kind of lipid metaboli activation lipoprotein receptors to inhibit the application in cell Hippo signal pathway activated.

Preferably, the application includes being prepared into have by lipid metaboli activation lipoprotein receptor to inhibit cell Hippo The drug of signal pathway activated function.

In the present invention, the lipid metaboli activation lipoprotein receptor, which preferably passes through, promotes the increased side of YAP protein phosphorylation Formula inhibits cell Hippo signal pathway activated.

The present invention provides a kind of lipid metaboli activation lipoprotein receptor answering in preparation prevention and/or treatment liver-cancer medicine With.

In the present invention, the lipid metaboli activation lipoprotein receptor preferably passes through the work for inhibiting normal cell Hippo access Change prevents liver cancer.

Alternatively, the lipid metaboli activation lipoprotein receptor preferably passes through the activation therapy for inhibiting liver cancer cells Hippo access Liver cancer.

The utility model has the advantages that the present invention provides a kind of lipid metaboli activation lipoprotein receptors, and YAP protein phosphorylation to be promoted to increase In application.The present invention the experiment proved that: lipid metaboli activates lipoprotein receptor physical efficiency that YAP protein phosphorylation is promoted to increase, and then makes YAP albumen is stranded in cytoplasm, to inhibit the Hippo signal pathway activated of cell, inhibits the proliferation of liver cancer cells and tumor formation big It is small, play the effect of prevention and/or treatment liver cancer.

Detailed description of the invention

Fig. 1 is that liver cancer cells Hep3B, Huh7 described in the embodiment of the present invention 1 strike low endogenous LSR rear clone formation (clone Formation) experimental result.

Fig. 2 is that liver cancer cells Hep3B, Huh7 described in the embodiment of the present invention 1 strike after low endogenous LSR that tumor formation is real in nude mouse Test result.

Fig. 3 is Western Blot testing result described in the embodiment of the present invention 2；

Fig. 4 is laser scanning co-focusing (confocal) experimental result described in the embodiment of the present invention 2；

Fig. 5 is cell caryoplasm separation test result described in the embodiment of the present invention 2；

Fig. 6 is co-immunoprecipitation experiment result described in the embodiment of the present invention 2；

Fig. 7 is the key of LSR protein structure schematic diagram described in the embodiment of the present invention 3, the effect of LSR and Hippo pathway protein Site and point mutation schematic diagram.

Fig. 8 is the co-immunoprecipitation result figure (Co-IP) of LSR and YAP albumen described in the embodiment of the present invention 3.

Fig. 9 is LSR (wild type and saltant type) described in the embodiment of the present invention 3 and YAP common location experimental result in the cell.

Figure 10 is real to the inhibiting effect western blot of Hippo access after being overexpression LSR described in the embodiment of the present invention 3 Test result.

Figure 11 is the co-immunoprecipitation experiment result of LSR (wild type and saltant type) and YAP described in the embodiment of the present invention 4 (Co-IP)。

Figure 12 is overexpression LSR (wild type and saltant type) described in the embodiment of the present invention 4 to Hippo access in liver cancer cells Inhibiting effect (western blot).

Figure 13 is the intracellular fixed altogether of overexpression LSR (wild type and saltant type) and YAP albumen described in the embodiment of the present invention 4 Position experimental result (confocal).

Specific embodiment

The present invention provides a kind of lipid metaboli activation lipoprotein receptors to promote the application in the increase of YAP protein phosphorylation. In the present invention, the amino acid sequence of the lipid metaboli activation lipoprotein receptor is as shown in SEQ ID No.5, and nucleotide sequence is such as Shown in SEQ ID No.6.Lipid metaboli activation lipoprotein receptor physical efficiency of the present invention passes through the site Y623 or the site S493 and YAP egg White combination, and then the phosphorylation of YAP albumen is promoted to increase.In the present invention, the application preferably includes to swash the lipid metaboli Lipoprotein receptor living is prepared into the drug for promoting YAP protein phosphorylation to increase function.The lipid metaboli activates lipoprotein receptor Body is to provide liver cancer treatment to provide possible therapy target.In the present invention, the albumen is in liver cancer as potential treatment Target spot, can by activation (reduction) its activity or increase (reductions) its expression, thus increase treat liver cancer feasible scheme or Person's therapy target.

The present invention provides a kind of lipid metaboli activation lipoprotein receptors to inhibit the application in cell Hippo signal pathway activated. Experiment shows that lipid metaboli activation lipoprotein receptor physical efficiency passes through the site Y623 or the site S493 and YAP protein binding, and then promotes The phosphorylation of YAP albumen increases.It after YAP protein phosphorylation increase, can be trapped in cytoplasm, and then can not be in Hippo access Downstream play activation.In the present invention, the application preferably includes to activate the lipid metaboli into lipoprotein receptor preparation At the therapy target with inhibition cell Hippo signal pathway activated function.In the present invention, the albumen is in liver cancer as potential Therapy target, can by activation (reduction) its activity or increase (reductions) its expression, thus increase treat liver cancer feasibility Scheme or therapy target.

The present invention also provides a kind of lipid metaboli activation lipoprotein receptors in preparation prevention and/or treatment liver-cancer medicine Using.Experiment shows that lipid metaboli activation lipoprotein receptor physical efficiency passes through the site Y623 or the site S493 and YAP protein binding, in turn The phosphorylation of YAP albumen is promoted to increase.It after YAP protein phosphorylation increase, can be trapped in cytoplasm, and then can not be in Hippo The downstream of access plays activation.For normal cell, inhibit normal cell using lipid metaboli activation lipoprotein receptor physical efficiency The activation of Hippo access, to play the role of preventing liver cancer；For liver cancer cells, lipoprotein receptor is activated using lipid metaboli It can inhibit the activation of liver cancer cells Hippo access, to play the role for the treatment of liver cancer.In the present invention, the albumen is in liver It is used as potential therapy target in cancer, by its activity of activation (reduction) or (reduction) its expression can be increased, to increase treatment The feasible scheme or therapy target of liver cancer.

YAP protein phosphatase is being promoted to a kind of lipid metaboli activation lipoprotein receptor provided by the invention below with reference to embodiment The application changed in increasing is described in detail, but they cannot be interpreted as limiting the scope of the present invention.

Embodiment 1

(1) cell culture: Hep3B, Huh7 hepatoma cell strain are chosen, the logarithmic proliferation phase is grown to.Wherein Hep3B cultivates item Part be+1% penicillin/streptomycin of+10% fetal calf serum of EMEM culture medium, Huh7 cell culture condition be DMEM culture medium+ + 1% penicillin/streptomycin (PS) of 10% fetal calf serum (FBS), 5%CO₂And it is cultivated under the conditions of 37 DEG C.

(2) stablize and strike low LSR cell strain building:

A. cell dissociation, counting, complete medium are suspended by 1d before testing, and are laid in six orifice plates, to cell fusion about 70- Virus infection is carried out when 80% (virus selects santa cruz's to strike low LSR virus liquid, article No.: sc-97082-V)；

B. after the virus liquid frozen being melted on ice, every hole addition 1ml virus liquid+1ml DMEM (10%FBS+P/S)+ 0.1% polybrene (polybrene) (4ug/ml)

C. 1ml DMEM (containing 10%FBS+P/S) is added in every hole after cultivating 12h

D. after cultivating for 24 hours, will be adherent after cell culture medium be changed to 2ml DMEM (containing 10%FBS+P/S)

E. dosing puromycin screens after infecting 48h；If cell covers with 6-Well, then will be under cell dissociation after infection for 24 hours Come, passage, which moves on in 10cm ware, to be continued to cultivate.

F. after cell is adherent, puromycin is added and carries out medicine sieve, the additive amount of puromycin is respectively as follows: Huh7 Puromycin 2ul/ml, Hep3B puromycin 1ul/ml, SNU449 puromycin 5ul/ml.It is thin after dosing 3-4d Born of the same parents initially enter peak mortality, after about 14d, macroscopic monoclonal cell strain occur.

F. after blank group complete cell death, the maintenance of half amount puromycin, after covering with 10cm culture dish, point six orifice plates are given Cell, cell protein sample to be done extract, and poor efficiency is struck in identification.

H. other cells expand, and give puromycin maintenance.

(3) cell strain for successfully striking low LSR is chosen, colony formation is carried out:

Cell culture grows to 80%-90% and merges and be in logarithmic growth phase in 10cm Dish.Hep3B and Huh7 Cell is collected cell after trypsin digestion and is counted.Take 6 orifice plates, diluting cells to suitable concentration, with the inoculation of 1000/hole It in 6 orifice plates, blows even, sets and cultivate 18h under 37 DEG C and 5%CO2 environment.Control group and experimental group are respectively provided with 3 multiple holes, stationary culture 2 weeks.When single clone cell group cell number occur in control group group group culture dish and being more than 50, culture is terminated.Discard culture Base, PBS are cleaned cell 2 times, and the fixed about 20min of 4% paraformaldehyde is added, discards later, and crystal violet dye liquor contaminates 20~30min, Then dyeing liquor is washed away with PBS, dried under room temperature.It takes pictures, microscopic counting is greater than clone's number of 50 cells.

Finally calculate cloning efficiency: cloning efficiency=clone formation number/inoculating cell number × 100%.Clone formation Rate result is as shown in Figure 1, Fig. 1 shows: after striking low Hep3B, Huh7 cell, Cell clonality enhancing.

(4) nude mice by subcutaneous tumor formation is tested:

It chooses 4 week old Female nude mice of SPF grade to be grouped at random, cuts ear tag note.Respectively by the control group of logarithmic growth phase with Experimental group cell dissociation, collection, counting, cell is resuspended in PBS, and final concentration of 2.0 × 10⁷/ ml, every needle 0.2ml inoculation The dorsal sc on the right side of nude mice.Nude mice, record nude mice state, weight, subcutaneous tumors length and width are observed every 3d.To control group There is termination when ulceration up to 1cm or tumor surface in subcutaneous tumors major diameter, and de- neck puts to death nude mice under anesthesia, takes subcutaneous tumors in 4% poly It is saved in formaldehyde.Gross tumor volume: V=0.5 × L × W is calculated by following publicity²(V: volume, L: major diameter, W: minor axis), statistics are each Group tumor size.Statistical result is as shown in Fig. 2, Fig. 2 shows: after striking low LSR, the enhancing of liver cancer cells nude mice one-tenth knurl ability.

The results showed that striking hepatoma cell proliferation ability after low LSR enhances, nude mice one-tenth knurl ability enhances the present embodiment.

Embodiment 2

(1) low LSR group and cellular control unit are struck in Western Blot detection, and grouping situation is control group, strikes low groups of cells 1, low groups of cells 2 is struck.

A. protein extraction

1. by grouping by cell point into culture dish, it is to be grown that (it is 90% that cell, which collects rate, in culture dish, at this time to 90% Cell is in logarithmic growth phase), remove cell culture fluid, is washed three times with PBS.

2. preparing protein lysate A (4 DEG C of preservations) in advance, the formula of protein lysate A is shown in Table 1, will using deionized water Volume is assigned to 500ml.

1 protein lysate A of table formula

Cell pyrolysis liquid B is prepared before lytic cell, the formula of cell pyrolysis liquid B is shown in Table 2

2 cell pyrolysis liquid B of table formula

Note: storing liquid refers to the concentration that initial concentration either reagent of formula raw material purchase usually stores.

Cell pyrolysis liquid B is added in Tissue Culture Dish.

3. lytic cell 3min on 4 DEG C of ice chests.

4. 1.5ml centrifuge tube under cell scraper, will be collected in scraper plate.

5. 4 DEG C of 15000 turns/min are centrifuged 10min, make pellet cell debris.Supernatant is total protein, extracts supernatant and moves into New pipe takes out 20ul and does protein quantification, and 6Xloading buffer is added in remaining, boils 6min, cooled on ice for 100 DEG C after mixing.

6. sealed membrane seals, -80 DEG C of preservations.

B. protein quantification

1. each protein sample is added in 96 orifice plates；

2. adding 1 volume BCA reagent B (50:1) to prepare appropriate BCA according to sample size by 50 volume BCA reagent As and working Liquid mixes well；

3. 200 μ l BCA working solutions are added in each hole；

30sec is vibrated on the oscillator 4. ELISA Plate is put, 37 DEG C of placement 30min, the then colorimetric estimation at 562nm. With protein content (μ g) for abscissa, light absorption value is ordinate, draws standard curve；

5. diluting sample to be tested to suitable concentration, make 20 μ l of sample diluting liquid total volume, addition 200 μ L of BCA working solution, It mixes well, after 37 DEG C of placement 30min, reference is done with No. 0 pipe of standard curve, the colorimetric under 562nm wavelength records light absorption value；

6. according to the light absorption value of institute's sample corresponding protein content (μ g) can be checked on standard curve, divided by sample Product dilution total volume (20 μ l) is sample actual concentrations (unit: μ g/ μ l) multiplied by sample extension rate.

C.SDS-PAGE electrophoresis

1. drying, it is spare putting into fixed bracket glass plate and comb tri-distilled water successively wash clean in oven.

2. installing vertical electrophoresis glass plate, 10% separation gel is prepared, is poured into glass plate gap after separation gel is mixed immediately In, upper layer covers isopropanol, and offset plate is disposed vertically at room temperature, and after glue polymerization completely to be separated, incline upper layer isopropanol, uses Filter paper blots.

3. preparing 4% concentration glue, directly it is poured on the separation gel solidified after concentration glue is mixed, is immediately inserted into comb Gel is disposed vertically and polymerize at room temperature by son.Glue is concentrated and separation gel is prepared, according to BIO-RAD company specification formula.

4. carefully extracting comb after glue polymerization completely to be concentrated, being fixed on electrophoretic apparatus, upper and lower slot is separately added into electrophoresis Buffer.

5. being loaded with micro sample adding appliance, 5 μ l of Marker, 50 μ g are added according to each well of protein quantification in remaining swimming lane Sample.Power on, glue 140V is concentrated；Separation gel 120V.Until bromophenol blue indicator reaches glue bottom, electrophoresis is terminated.By glue Electrotransfer is to cellulose acetate film (NC film).

D. transferring film and protein staining

After electrophoresis, separation gel and an equal amount of NC film and 2 filter paper are immersed in transfering buffering liquid, equilibrium at room temperature 30min.Separation gel is close to film, 1 filter paper is respectively sticked in two sides, and edge alignment is driven away bubble, is clipped in sponge, separation gel Close to negative side, film is packed into electrotransfer slot at anode.Jump condition is voltage 120v constant pressure, is determined and is turned according to molecular weight The film time takes out film, and marks.After electrotransfer, film is dyed with Ponceaux, and labelled protein size simultaneously cuts film.

E. milk powder closing and antibody incubation

1. film is placed in 5% skimmed milk power, room temperature closes 2-3h.

2. film is placed in the primary antibody that corresponding proportion has diluted, 4 DEG C of overnight incubations.After next day room temperature restores 1h, TBST is washed Film 3 times, each 10min.

3. film is placed in the secondary antibody that the corresponding corresponding proportion of primary antibody has diluted, room temperature 1h.Film 3 times are washed with TBST, every time 10min。

4. ECL shines: A, B luminescent solution 1:1 are diluted and mixed, avoid light place 1min.Film is slightly rinsed with PBS, and filter paper is inhaled It is dry, by A, B mixing drop on film.It shines using BIO-RAD light-emitting appearance, same albumen, fixed fluorescent lifetime, and demarcate Marker is analyzed.

F. result judgement

Protein blot and each experiment of RT-PCR are repeated 3 times and do gray analysis, are as a result indicated with mean ± standard deviation.And Take group difference that the one-way analysis of variance in 17.0 software of SPSS and t is used to examine.It is to have conspicuousness poor with p < 0.05 Different, p < 0.01 is to have extremely significant difference.

WesternBlot testing result is as shown in figure 3, Fig. 3 is shown: after Hep3B, Huh7 cytotostatic strike low LSR, YAP Protein phosphorylation is reduced, and downstream CTGF, Cyr61 protein expression rises.The result shows that: Hippo access core egg can be made by striking low LSR White YAP phosphorylation reduces, and YAP albumen enters core increase, and downstream protein expression increases.

(2) laser scanning co-focusing (confocal) is tested

A. cell count divides cell.

Prepare new Tissue Culture Dish, creep plate is put into culture dish, is soaked with culture medium；It is grouped, will count according in figure Several points of good cells are taped against in culture dish after mixing, and cultivate cell 16-18h.After 24-48h cell is adherent, creep plate is made.

1. preparing paraformaldehyde (PFA): the m- 20 DEG C of refrigerators of cell take out defrosting to solution without ice in advance；

2. cell is taken out from 37 DEG C of incubators, softly washed 3 times with the PBS of pre-temperature；

3. going in six new orifice plates/35mm ware, notice that the cell of creep plate faces upward, with 4 DEG C of paraformaldehyde, 2ml/ Hole, the fixed 45min of room temperature；

4. being washed three times with PBS, method is same as above after fixed；

5. 0.2%Triton X-100 permeabilization 20min；PBS is washed three times, ibid；

6. the BSA with 3% closes 1h, the hole 2ml/；

7. being washed three times after closing with PBS, it is proportionally added into primary antibody, 4 DEG C overnight；

8. after overnight, being protected from light and being washed cell 3 times with the PBS of pre-temperature is light and slow；

The diluted secondary antibody of 3%BSA is used 9. being added, is protected from light incubation at room temperature 1h；

10. being washed 3 times, being kept in dark place with PBS in darkroom after being incubated for；

B. creep plate is done in darkroom.

It is dried 1. in advance washing glass slide

2. the cell in PBS is taken out, cell is face-down, covers on glass slide, and a small amount of anti-quencher is added.

3. Confocal observes cell.

Laser scanning co-focusing (confocal) experimental result is as shown in Figure 4.Fig. 4 shows Hep3B, Huh7 cell strain laser The burnt display YAP of copolymerization (green 488), nucleus are blue (DAPI): liver cancer cells stabilization strikes YAP albumen nuclear location after low LSR Increase.

(3) cell caryoplasm separation test

1. digesting, cell is counted, spreads back cell in culture dish again by grouping.

NP-40 lysis buffer is prepared by table 3,

Table 3:NP-40 lysis buffer formula

NP-40 lysis buffer	to prepare 100mL
		0.5%Nonidet P-40	500ul
20mM Tris/HCl, pH7.5	2mL 1M Tris-Hcl pH7.5
		100mM NaCl	2mL 5M NaCl 0.5844g
50mM NaF	5mL 1M NaF 0.20995g

2. 10cm culture dish PBS is washed 2 times, add the hole PBS700ul/, cell scraper to 1.5ml EP is managed

3. 4 DEG C of 300g*5min abandon supernatant

4. (PMSF, NaVO4 is added in above-mentioned lysate B ratio) in NP-40,400ul/ pipe is dispelled

⑤4°10000g*5min

6. drawing supernatant does cytoplasm protein

7. 110 DEG C of precipitating plus 1%SDS 100ul/ pipe boil, it is vortexed, 10min is nucleoprotein

8. doing standard curve (NP-40, SDS) respectively with two kinds of lysates, protein concentration is surveyed by above-mentioned BCA method,

9. calculating protein concentration, adjustment protein concentration to same concentrations.6Xloading is added, boils sample, after cooled on ice, Western blot is carried out by above-mentioned steps.

As shown in figure 5, Fig. 5 shows to strike after low LSR, expression quantity increases cell caryoplasm separation test result in the nucleus of YAP Add, content declines in cytoplasm, the decline of cytoplasm p-YAP phosphorylation.

(4) co-immunoprecipitation experiment.

It is to be transfected in morning next day (to use after 16-18h 1. cell dissociation is laid in 6cm culture dish by being grouped in figure Lipo2000), complete medium is replaced after having transfected 5.5-6h, incubation is tested for 24 hours.

2. preparing lysate B, take cell on ice, mark, abandon culture solution, PBS is washed 2 times, and vacuum blots.Each ware adds Enter 500ul Lysis, stands 5min on ice.

3. being wiped off cell in ware with cell scraper, cell cracking suspension is drawn into EP pipe, and oscillation mixes, and stands on ice 25min.After having stood, 4 DEG C of centrifugations, 15000rpm × 10min.

4. supernatant is drawn onto the EP pipe of new pre-cooling.In addition 70ul supernatant is drawn into another group of EP pipe, and 14ul is added 6 × Loading Buffer (5:1), boils 5min, for doing the control of albumen sample.

5. remaining one group of EP manages every pipe and primary antibody is added, is sealed, be fixed on blending instrument with sealed membrane, 4 DEG C of mixing 1h.

6. 20min washes Protein G with PBS in advance, after mixing pearl, 40ul is vacantly added in every pipe.EP pipe is fixed on In 50ml centrifuge tube, 4 DEG C slowly shake up 2.5h.

7. preparing washing lotion (50ml centrifuge tube) by lotion prescription shown in table 4:

Table 4: lotion prescription

8. removing sealed membrane, 4 DEG C of centrifugations, 10000rpm × 1min abandons supernatant.1ml washing lotion is washed, and 5 times, each 10000rpm ×1min。

9. blotting per effective vacuum pump, every pipe adds 1 × Loading of 40ul Buffer, 8000rpm × 1min.It boils 6min, ice bath cooling.4 DEG C, 8000rpm × 1min centrifugation, supernatant is transferred in new EP pipe.By abovementioned steps western blot。

Co-immunoprecipitation experiment result as shown in fig. 6, Fig. 6 the result shows that: the combination of LSR and YAP subtracts in upstream activator Few, by inhibiting upstream kinases activity, the combination of LSR and YAP are restored

Embodiment 3

(1) (LSR plasmid is granted by Dalian Medical Univ professor Meng Songshu, and provides its protein sequence), LSR structural diagrams And mutational site is as shown in Figure 7.It is step by step rapid to carry out co-immunoprecipitation, with 2 step of embodiment (4), is grouped as shown in Figure 8, wherein Primary antibody is anti-myc antibody (myc-invitrogen/132500, by specification ratio are added), co-immunoprecipitation result such as Fig. 8 institute Show, Fig. 8 shows: external LSR and YAP albumen have interaction, and LSR saltant type and YAP are without interaction.

(2) implement copolymerization coke by preceding method, be grouped as shown in Figure 9, laser co-focusing result is as shown in Figure 9, wherein YAP (green), LSR (red), nucleus are blue (DAPI)；Fig. 9 shows: in COS7 cell, YAP and LSR are in the cell There is common location.

(3) construct overexpressing cell strain (carrier be pCDH- Lentiviral, packaging plasmid psPAX2, PMD2.G), including be overexpressed LSR-WT, LSR-YA hepatoma cell strain (LSR-WT plasmid from Dalian Medical Univ Meng pine tree religion Award), construction method is referring to " Molecular Cloning:A Laboratory guide ".

Connect over-express vector primer: upstream is cggaattcgccaccatgcaacaggacggacttggagtag (SEQ ID No.1)；Downstream is cgggatccctacgtagaatcgagaccgaggagagg (SEQ ID No.2).

Construct LSR mutant；LSR-YA upstream primer: cccgcccgcgccgcccccggcctcggagaccg (SEQ ID No.3)；LSR-YA (downstream) primer: gcctgcgagtcggtctccgaggccgggggcggc (SEQ ID No.4).

(4) it such as aforementioned western experimental procedure, is grouped by Figure 10, Western blot testing result such as Figure 10 institute Show, Figure 10 shows: inhibiting Hippo passage downstream protein expression after being overexpressed LSR-WT, and be overexpressed mutant LSR-YA aftereffect It should be weakened.

This example demonstrates that: LSR can influence its combination by being mutated its binding site in conjunction with YAP protein-specific Intensity.Being overexpressed LSR can be such that YAP phosphorylation increases, and YAP albumen enters to examine and make cuts less, the decline of downstream protein expression.

Embodiment 4

(1) it is co-precipitated experimental procedure by foregoing immune, is grouped by Figure 11, co-immunoprecipitation experiment result such as Figure 11 institute Show, Figure 11 the result shows that: the interaction of LSR-SA mutant and YAP are weak relative to LST-WT.

(2) overexpressing cell strain (method is with embodiment 3) is constructed, is overexpressed LSR-SA hepatoma cell strain.

(3) Western blot experiment is carried out by abovementioned steps, is grouped by Figure 12, Western blot result is as schemed Shown in 12, Figure 12 the result shows that: be overexpressed LSR-SA after equally make YAP phosphorylation increase, but than wild type act on it is weak.

(4) laser co-focusing test is carried out by abovementioned steps, is grouped by Figure 13, laser co-focusing test result is as schemed Shown in 13, Figure 13 the result shows that: be overexpressed LSR-WT, LSR-SA (red) after, YAP (green) apoptotic nueleolus reduction, mistake table Up to after LSR-YA, effect restores.

This example demonstrates that: LSR is related with 14-3-3 to the effect of YAP albumen, and the site LSR-S493 is LSR and 14-3-3 Binding site, the LSR-S493A after mutation cannot be with 14-3-3 protein binding (existing document report), and 14-3-3 albumen is Hippo signal path important composition albumen.

The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered It is considered as protection scope of the present invention.

Sequence table

<110>Shenzhen University

<120>a kind of lipid metaboli activation lipoprotein receptor is promoting the application in the increase of YAP protein phosphorylation

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<170> SIPOSequenceListing 1.0

<210> 1

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<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 1

cggaattcgc caccatgcaa caggacggac ttggagtag 39

<210> 2

<211> 35

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 2

cgggatccct acgtagaatc gagaccgagg agagg 35

<210> 3

<211> 32

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 3

cccgcccgcg ccgcccccgg cctcggagac cg 32

<210> 4

<211> 33

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 4

gcctgcgagt cggtctccga ggccgggggc ggc 33

<210> 5

<211> 649

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 5

Met Gln Gln Asp Gly Leu Gly Val Gly Thr Arg Asn Gly Ser Gly Lys

1 5 10 15

Gly Arg Ser Val His Pro Ser Trp Pro Trp Cys Ala Pro Arg Pro Leu

20 25 30

Arg Tyr Phe Gly Arg Asp Ala Arg Ala Arg Arg Ala Gln Thr Ala Ala

35 40 45

Met Ala Leu Leu Ala Gly Gly Leu Ser Arg Gly Leu Gly Ser His Pro

50 55 60

Ala Ala Ala Gly Arg Asp Ala Val Val Phe Val Trp Leu Leu Leu Ser

65 70 75 80

Thr Trp Cys Thr Ala Pro Ala Arg Ala Ile Gln Val Thr Val Ser Asn

85 90 95

Pro Tyr His Val Val Ile Leu Phe Gln Pro Val Thr Leu Pro Cys Thr

100 105 110

Tyr Gln Met Thr Ser Thr Pro Thr Gln Pro Ile Val Ile Trp Lys Tyr

115 120 125

Lys Ser Phe Cys Arg Asp Arg Ile Ala Asp Ala Phe Ser Pro Ala Ser

130 135 140

Val Asp Asn Gln Leu Asn Ala Gln Leu Ala Ala Gly Asn Pro Gly Tyr

145 150 155 160

Asn Pro Tyr Val Glu Cys Gln Asp Ser Val Arg Thr Val Arg Val Val

165 170 175

Ala Thr Lys Gln Gly Asn Ala Val Thr Leu Gly Asp Tyr Tyr Gln Gly

180 185 190

Arg Arg Ile Thr Ile Thr Gly Asn Ala Asp Leu Thr Phe Asp Gln Thr

195 200 205

Ala Trp Gly Asp Ser Gly Val Tyr Tyr Cys Ser Val Val Ser Ala Gln

210 215 220

Asp Leu Gln Gly Asn Asn Glu Ala Tyr Ala Glu Leu Ile Val Leu Gly

225 230 235 240

Arg Thr Ser Gly Val Ala Glu Leu Leu Pro Gly Phe Gln Ala Gly Pro

245 250 255

Ile Glu Asp Trp Leu Phe Val Val Val Val Cys Leu Ala Ala Phe Leu

260 265 270

Ile Phe Leu Leu Leu Gly Ile Cys Trp Cys Gln Cys Cys Pro His Thr

275 280 285

Cys Cys Cys Tyr Val Arg Cys Pro Cys Cys Pro Asp Lys Cys Cys Cys

290 295 300

Pro Glu Ala Leu Tyr Ala Ala Gly Lys Ala Ala Thr Ser Gly Val Pro

305 310 315 320

Ser Ile Tyr Ala Pro Ser Thr Tyr Ala His Leu Ser Pro Ala Lys Thr

325 330 335

Pro Pro Pro Pro Ala Met Ile Pro Met Gly Pro Ala Tyr Asn Gly Tyr

340 345 350

Pro Gly Gly Tyr Pro Gly Asp Val Asp Arg Ser Ser Ser Ala Gly Gly

355 360 365

Gln Gly Ser Tyr Val Pro Leu Leu Arg Asp Thr Asp Ser Ser Val Ala

370 375 380

Ser Glu Val Arg Ser Gly Tyr Arg Ile Gln Ala Ser Gln Gln Asp Asp

385 390 395 400

Ser Met Arg Val Leu Tyr Tyr Met Glu Lys Glu Leu Ala Asn Phe Asp

405 410 415

Pro Ser Arg Pro Gly Pro Pro Ser Gly Arg Val Glu Arg Ala Met Ser

420 425 430

Glu Val Thr Ser Leu His Glu Asp Asp Trp Arg Ser Arg Pro Ser Arg

435 440 445

Gly Pro Ala Leu Thr Pro Ile Arg Asp Glu Glu Trp Gly Gly His Ser

450 455 460

Pro Arg Ser Pro Arg Gly Trp Asp Gln Glu Pro Ala Arg Glu Gln Ala

465 470 475 480

Gly Gly Gly Trp Arg Ala Arg Arg Pro Arg Ala Arg Ser Val Asp Ala

485 490 495

Leu Asp Asp Leu Thr Pro Pro Ser Thr Ala Glu Ser Gly Ser Arg Ser

500 505 510

Pro Thr Ser Asn Gly Gly Arg Ser Arg Ala Tyr Met Pro Pro Arg Ser

515 520 525

Arg Ser Arg Asp Asp Leu Tyr Asp Gln Asp Asp Ser Arg Asp Phe Pro

530 535 540

Arg Ser Arg Asp Pro His Tyr Asp Asp Phe Arg Ser Arg Glu Arg Pro

545 550 555 560

Pro Ala Asp Pro Arg Ser His His His Arg Thr Arg Asp Pro Arg Asp

565 570 575

Asn Gly Ser Arg Ser Gly Asp Leu Pro Tyr Asp Gly Arg Leu Leu Glu

580 585 590

Glu Ala Val Arg Lys Lys Gly Ser Glu Glu Arg Arg Arg Pro His Lys

595 600 605

Glu Glu Glu Glu Glu Ala Tyr Tyr Pro Pro Ala Pro Pro Pro Tyr Ser

610 615 620

Glu Thr Asp Ser Gln Ala Ser Arg Glu Arg Arg Leu Lys Lys Asn Leu

625 630 635 640

Ala Leu Ser Arg Glu Ser Leu Val Val

645

<210> 7

<211> 1950

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 7

atgcaacagg acggacttgg agtagggaca aggaacggaa gtgggaaggg gaggagcgtg 60

cacccctcct ggccttggtg cgcgccgcgc cccctaaggt actttggaag ggacgcgcgg 120

gccagacgcg cccagacggc cgcgatggcg ctgttggccg gcgggctctc cagagggctg 180

ggctcccacc cggccgccgc aggccgggac gcggtcgtct tcgtgtggct tctgcttagc 240

acctggtgca cagctcctgc cagggccatc caggtgaccg tgtccaaccc ctaccacgtg 300

gtgatcctct tccagcctgt gaccctgccc tgtacctacc agatgacctc gacccccacg 360

caacccatcg tcatctggaa gtacaagtct ttctgccggg accgcatcgc cgatgccttc 420

tccccggcca gcgtcgacaa ccagctcaat gcccagctgg cagccgggaa cccaggctac 480

aacccctacg ttgagtgcca ggacagcgtg cgcaccgtca gggtcgtggc caccaagcag 540

ggcaacgctg tgaccctggg agattactac cagggccgga ggattaccat caccggaaat 600

gctgacctga cctttgacca gacggcgtgg ggggacagtg gtgtgtatta ctgctccgtg 660

gtctcagccc aggacctcca ggggaacaat gaggcctacg cagagctcat cgtccttggg 720

aggacctcag gggtggctga gctcttacct ggttttcagg cggggcccat agaagactgg 780

ctcttcgtgg ttgtggtatg cctggctgcc ttcctcatct tcctcctcct gggcatctgc 840

tggtgccagt gctgcccgca cacttgctgc tgctacgtca ggtgcccctg ctgcccagac 900

aagtgctgct gccccgaggc cctgtatgcc gccggcaaag cagccacctc aggtgttccc 960

agcatttatg cccccagcac ctatgcccac ctgtctcccg ccaagacccc acccccacca 1020

gctatgattc ccatgggccc tgcctacaac gggtaccctg gaggataccc tggagacgtt 1080

gacaggagta gctcagctgg tggccaaggc tcctatgtac ccctgcttcg ggacacggac 1140

agcagtgtgg cctctgaagt ccgcagtggc tacaggattc aggccagcca gcaggacgac 1200

tccatgcggg tcctgtacta catggagaag gagctggcca acttcgaccc ttctcgacct 1260

ggccccccca gtggccgtgt ggagcgggcc atgagtgaag tcacctccct ccacgaggac 1320

gactggcgat ctcggccttc ccggggccct gccctcaccc cgatccggga tgaggagtgg 1380

ggtggccact ccccccggag tcccagggga tgggaccagg agcccgccag ggagcaggca 1440

ggcgggggct ggcgggccag gcggccccgg gcccgctccg tggacgccct ggacgacctc 1500

accccgccga gcaccgccga gtcagggagc aggtctccca cgagtaatgg tgggagaagc 1560

cgggcctaca tgcccccgcg gagccgcagc cgggacgacc tctatgacca agacgactcg 1620

agggacttcc cacgctcccg ggacccccac tacgacgact tcaggtctcg ggagcgccct 1680

cctgccgacc ccaggtccca ccaccaccgt acccgggacc ctcgggacaa cggctccagg 1740

tccggggacc tcccctatga tgggcggcta ctggaggagg ctgtgaggaa gaaggggtcg 1800

gaggagagga ggagacccca caaggaggag gaggaagagg cctactaccc gcccgcgccg 1860

cccccgtact cggagaccga ctcgcaggcg tcccgagagc gcaggctcaa gaagaacttg 1920

gccctgagtc gggaaagttt agtcgtctga 1950

Claims (10)

1. a kind of lipid metaboli activation lipoprotein receptor is promoting the application in the increase of YAP protein phosphorylation.

2. application according to claim 1, which is characterized in that being prepared into lipid metaboli activation lipoprotein receptor has YAP protein phosphorylation is promoted to increase the drug of function.

3. application according to claim 1, which is characterized in that the lipid metaboli activation lipoprotein receptor passes through the site Y623 YAP protein phosphorylation is promoted to increase with the protein bound mode of YAP.

4. application according to claim 1, which is characterized in that the lipid metaboli activation lipoprotein receptor passes through the site S493 YAP protein phosphorylation is promoted to increase with the protein bound mode of YAP.

5. a kind of lipid metaboli activation lipoprotein receptor is inhibiting the application in cell Hippo signal pathway activated.

6. application according to claim 5, which is characterized in that being prepared into lipid metaboli activation lipoprotein receptor has Inhibit the drug of cell Hippo signal pathway activated function.

7. application according to claim 5, which is characterized in that the lipid metaboli activation lipoprotein receptor is by promoting YAP The increased mode of protein phosphorylation inhibits cell Hippo signal pathway activated.

8. a kind of application of lipid metaboli activation lipoprotein receptor in preparation prevention and/or treatment liver-cancer medicine.

9. application according to claim 8, which is characterized in that the lipid metaboli activation lipoprotein receptor is by inhibiting normal The activation of cell Hippo access prevents liver cancer.

10. application according to claim 8, which is characterized in that the lipid metaboli activation lipoprotein receptor is by inhibiting liver The activation therapy liver cancer of cancer cell Hippo access.