CN107525932A - Applications of the SH3BGRL and its mRNA in the diagnostic kit or medicine for preparing breast cancer - Google Patents

Abstract

The invention discloses applications of the SH3BGRL and its mRNA in the diagnostic kit or medicine for preparing breast cancer, the SH3BGRL and SH3BGRL itself of a large amount expression physicochemical property can be as the labels of breast cancer clinical diagnosis and prognosis；The signal transduction pathway of SH3BGRL mediations is disturbed, breast cancer caused by the high expression of SH3BGRL can be treated.The research of the present invention finds that the preparation to breast cancer clinical diagnosis, prognosis and medicine has facilitation.

Description

SH3BGRL and its mRNA is in the diagnostic kit or medicine for preparing breast cancer Application

Technical field

The invention belongs to biomedicine technical field, more particularly to SH3BGRL and its mRNA to prepare the diagnosis of breast cancer Application in kit and medicine.

Background technology

SH3BGRL (SH3domain binding glutamic acid-rich protein like protein, SH3 Domain combines glutamic acid Abundant protein sample albumen) found first from Down's syndrome sufferer, positioned at No. 21 chromosome On.SH3BGRL does not have the expression specificity of organ-tissue, generally in marrow, liver, heart, kidney, brain, lung and placenta etc. just Expressed in normal histoorgan.SH3BGRL and SH3BGR, SH3BGRL2 and SH3BGRL3 have quite similar amino acid homology Property, thioredoxin is belonged to, is classified as SH3BG protein families.

China scientist reports its crystal structure first, and structural analysis shows that SH3BGRL includes SH3 and EVH1 two Amino acid region (domain), SH3BGRL belong to connection albumen (scaffold protein) group.In cell, containing SH3 The connection albumen in region can generally connect the protein molecular for including the region that can be combined with each other in unlike signal pipeline Come, so as to which unlike signal conduction path interweave with getting up, participate in signal transduction and gene expression regulation, such as：Participate in cellular elements Signal transduction, cytoskeleton rearrangement (cytoskeleton rearrangement) and cell membrane transporter (membrane The main cell metabolic pathway such as traffick).If the expression regulation of this albuminoid is affected, body disease will be caused, such as Cancer occurs and transfer.For example, the generation and transfer in tumour such as Grb2,14-3-3, mda-9/Syntenin and p130Cas During play highly important role.

There is a report to show SH3BGRL3s of the SH3BGRL with family obvious up-regulated expressions in bladder transitional cell carcinoma recently, And it is proportionate (Chiang CY, Pan CC, Chang HY, et al. SH3BGRL3protein with malignancy as a potential prognostic biomarker for urothelial carcinoma:a novel binding partner of epidermal growth factor receptor.Clin Cancer Res,2015,21: 5601- 5611.).The analysis of clinical disease sample shows SH3BGRL and Down's syndrome (Egeo A, Mazzocco M, Arrigo P, et indirectly al.Identification and characterization of a new human gene encoding a small protein with high homology to the proline-rich region of the SH3BGR gene. Biochemical and Biophysical Research Communications,1998,247:302-306), Parkinson Disease (Werner CJ, Heyny-von Haussen, R, Mall G, et al.Proteome analysis of human Substantia nigra in Parkinson ' s disease.Proteome Science, 2008,6-8.) it is related；Swollen In terms of transfer occurs for knurl, have and first report that display SH3BGRL can suppress the cell carcinogenesis as caused by viral gene v-Rel and turn Change (Majid SM, Liss AS, You M, et al.The suppression of SH3BGRL is important for v-Rel-mediated transformation.Oncogene,2006,25:756-768.).In addition, have been reported that display SH3BGRL is tumor suppressor gene (Wang H, Liu B, Al-aidaroos AQ, Li L, et al.Dual-faced SH3BGRL:oncogenic in mice,tumor suppressive in humans. Oncogene,2016,35:3303- 3313.)。

Breast cancer has turned into the disease problem of today's society, and the clinical diagnosis and prognosis of early stage are advantageous to controlling for breast cancer Treat, while the medicine of breast cancer is up for further research and development.SH3BGRL is not yet clear bright in the mechanism of action of breast cancer .

The content of the invention

Present invention aims to overcome that the shortcomings of the prior art, and SH3BGRL and its mRNA are provided and preparing mammary gland Application in the diagnostic kit or medicine of cancer.To achieve the above object, the technical scheme taken of the present invention is： Applications of the SH3BGRL or SH3BGRL mRNA in screening or preparing the kit of breast cancer clinical diagnosis or prognosis.

In addition, the reagent that the present invention also provides detection SH3BGRL or SH3BGRL mRNA is examined in preparation breast cancer clinic Application in the disconnected or kit of prognosis.

In addition, the present invention also provides SH3BGRL genes, SH3BGRL mRNA or SH3BGRL are screening or prepared treatment Application in the medicine of breast cancer.

In addition, the present invention also provides application of the SH3BGRL inhibitor in the medicine for preparing treatment breast cancer.

As the improvement of above-mentioned technical proposal, the SH3BGRL inhibitor includes suppressing SH3BGRL mRNA translations SiRNA or the antibody specifically bound with SH3BGRL.

In addition, the present invention also provides a kind of kit of breast cancer clinical diagnosis or prognosis, the kit includes measure The specific primer of SH3BGRL mRNA transcription amounts, determine the antibody or detection SH3BGRL physicochemical properties of SH3BGRL translation amounts Reagent, the albumen and the isoelectric point of itself, molecular weight that the SH3BGRL is combined changes.

In addition, the present invention also provides the medicine for the treatment of breast cancer, the medicine includes SH3BGRL inhibitor.

As the improvement of above-mentioned technical proposal, the SH3BGRL inhibitor includes suppressing SH3BGRL mRNA translations SiRNA or the antibody specifically bound with SH3BGRL.

The beneficial effects of the present invention are：The present invention provides SH3BGRL and its mRNA and is preparing the diagnostic reagent of breast cancer Application in box or medicine, the present invention is analyzed by gene data, Western blotting and immunochemical assays are found SH3BGRL genes high expression (including transcription and translation) in breast cancer tissue；Co-IP (co-immunoprecipitation) experiments are found SH3BGRL and Her2 (Epidermal growth factor-recepor-2) effect of be combineding with each other, can activate breast cancer cell downstream signal and lead to Road, promote breast cancer cell in Mice Body into knurl；The analysis of clinical data finds, the patient with breast cancer of SH3BGRL low expressions With higher survival rate and longer time-to-live；The combination of co-immunoprecipitation and two dimensional gel electrophore- sis experiment (IP-2D) hair In existing breast cancer cell there is change in the albumen of SH3BGRL combinations and the isoelectric point of itself and molecular size range；The present invention's Existing experiment fully shows：1) SH3BGRL expression and the protein modified state of itself can be used as breast cancer clinical diagnosis And the mark of prognosis；2) by the signal transduction pathway for disturbing SH3BGRL to mediate, the high expression of SH3BGRL can be treated and caused Breast cancer, brand-new as one SH3BGRL drug target treats breast cancer.

Brief description of the drawings

Fig. 1 shows the expression of SH3BGRL in the normal galactophore tissue of the embodiment of the present invention 1 and breast cancer tissue；Figure 1A is shown SH3BGRL expression quantity in the epithelial cell of normal galactophore tissue and lobular hyperplasia breast tissue, wherein, * represents p<0.05, n =8 (GEO：GDS2739)；SH3BGRL expression in the cell of Figure 1B display normal galactophore tissues and aggressive breast cancer tissue Amount, wherein, * represents p<0.05, n=6 (GEO： GDS4114)；Fig. 1 C show triple negative breast cancer tissue and non-three negative breast SH3BGRL expression quantity in the cell of cancerous tissue, wherein, * represents p<0.05, n=5,14, using t check analysis methods (GEO： GDS4069/8168557)；Fig. 1 D show SH3BGRL eggs in normal breast cancer beside organism (N) and breast cancer tissue (T) White expression quantity；Fig. 1 E show SH3BGRL expressing quantities in normal galactophore tissue and breast cancer tissue.

Fig. 2 shows that SH3BGRL is combined activation downstream signaling pathway with Her2 in the embodiment of the present invention 2, promotes breast cancer thin Born of the same parents are in Mice Body into knurl；Fig. 2A show the SH3BGRL of overexpression in non-triple negative breast cancer cell can activate PI3K and AKT paths；Fig. 2 B show that the SH3BGRL of overexpression can promote non-triple negative breast cancer cell in Mice Body into knurl；Figure 2C shows that SH3BGRL and Her2 interacts in 293T cells；

Fig. 3 shows the survival rate of patient with breast cancer and time-to-live in the embodiment of the present invention 3；

Fig. 4 shows that the albumen that the SH3BGRL of tumor tissue cell in the embodiment of the present invention 4 is combined changes；In figure Ellipse marks the albumen of SH3BGRL combinations；

Fig. 5 shows that the SH3BGRL of tumor tissue cell isoelectric point and molecular weight change in the embodiment of the present invention 4； Ellipse in figure marks SH3BGRL dielectrophoresis site.

Embodiment

For the object, technical solutions and advantages of the present invention are better described, below in conjunction with specific embodiment, subordinate list and The invention will be further described for accompanying drawing.

The measure of embodiment 1SH3BGRL expression quantity

Data analysis

From GEO：GDS database obtains relevant information, to the SH3BGRL in normal galactophore tissue and breast cancer tissue MRNA transcription amounts are analyzed, as shown in figs. 1 a to 1 c.

Immunoblot experiment

1) protein sample extracts：After culture medium in the culture dish for cultivating breast cancer cell is absorbed totally, according to culture dish The appropriate RIPA lysates of how much additions of middle cell concentration, cell is scraped from culture dish with cell scraper；By cell pyrolysis liquid It is collected into 1.5ml EP pipes, stands 30min on ice；4 DEG C of centrifugation 15min, supernatant are carefully drawn onto in new EP pipes, noted Meaning not be drawn onto precipitation；By the 5 × SDS-Loading Buffer solution prepared in advance by 3：1 ratio adds β-sulfydryl second Alcohol, by volume example be added in above-mentioned supernatant and fully mix；EP pipes are placed in 100 DEG C of heating 5min in dry-type thermostat Denaturation, short-term preservation are put in 4 DEG C, are put in for a long time in -20 DEG C.

2) encapsulating and loading：The glass plate for cleaning up and having dried is inserted into glue neck, makes bottom smooth, Preventing from cementing leakage, high one side is inwardly；15%SDS- polyacrylamide gel electrophoresis solution is prepared, separation gel, gelling to be separated Gu after good (about 30min), then 4% concentration glue；After 20min, carefully pull up comb, sequentially add 3 μ l marker and 10mg protein examples, careful sample overflows well and pollutes other ducts during sample-adding, and liquid-transfering gun gently leans against well during sample-adding On the glass plate of top, then slowly it is loaded, prevents from going out；Lid is covered tightly, notices that both positive and negative polarity is corresponding, adjusts voltage, concentrate glue 45V, 20~25min；Separation gel 90V, until bromophenol blue runs out of separation gel, about 2h.

3) transferring film：The complete gel of electrophoresis is removed, scrapes off concentration glue part, then by gel neatly on being laid in State on ready " sandwich ", pay attention to sampfe order and sample-adding during order transferring film on the contrary, rolling bubble, then pressed from both sides with tweezers Take pvdf membrane to be laid on gel, roll bubble, then spread filter paper successively, foam-rubber cushion, then clamp and be put into electrophoresis tank (transferring film It is special), black is to electrophoresis tank negative pole (black), and white/transparent side is to electrophoresis tank positive pole (red)；Put in electrophoresis tank side Enter ice chest, cover lid, electrophoresis tank is integrally put into foam box, then add ice cube and embedded, adjust voltage 90V, turn 2~3h of film (regulation voltage 25V, 4~5h of transferring film is overnight)；As electric current is excessive, constant current mode can be used：Calculation formula：Every square Cm x 2mA.

4) close：Trace is put into 5% skimmed milk power confining liquid up, and 1~2h (rotating speeds are incubated on decolorization swinging table It is adjusted at a slow speed).

5) immune response

It is incubated primary antibody：After the completion of closing, band where detecting albumen needed for the neat clip of tweezers, and with pencil mark Remember positive and negative and band title or indications, then trace is laid in the lid for posting sealed membrane up, and addition is matched somebody with somebody in advance Good primary antibody (- 20 DEG C of preservations), is incubated overnight, antibody recovery；Wash film：Pvdf membrane is taken out, is put into TBS-T liquid, is decolourizing to shake Room temperature elution 3~5 times on bed, each 10min, it is most importantly when washing film within a certain period of time (1~2h), number is tried one's best more； It is incubated secondary antibody：Pvdf membrane is taken out, secondary antibody to be incubated, the secondary antibody diluted in right amount with confining liquid is drawn with liquid-transfering gun, is added drop-wise to Pvdf membrane surface, is covered, and is then placed within incubation (most at a slow speed) 1.5~2.5h, each bar on room temperature decolorization swinging table With the two corresponding anti-solution added typically in 100~300 μ l, it should check whether pvdf membrane is dried frequently during this period, and add two It is anti-；Wash film：After secondary antibody has been incubated, equally eluted 5 times with PBST, each 10min.

6) chromogenic reaction：Substrate reactions liquid is prepared, strengthens luminous agent：Stabilizer=1：1,0.5~2.0 ml is typically prepared, Notice that substrate reactions liquid answers lucifuge, fail for 8h；Band is taken out and is laid in by albumen size order and posts sealed membrane On lid, the marking is face-up, then draws reaction solution cover layer surface with liquid-transfering gun, reacts 2~3min.

7) develop：After reacting 2~3min, one jiao of pvdf membrane is gripped with tweezers, unnecessary reaction solution is drawn on filter paper, so Neatly it to be put on the plastic sheeting in clamping plate afterwards, the marking is face-up, covers clamping plate, and is gently wiped on film with paper handkerchief, Rush wherein unnecessary reaction solution and bubble；An X film is carefully taken out in darkroom, is laid on pvdf membrane relevant position, It is sure not to move after spreading, the time for exposure can be from 1 second to a few hours, depending on fluorescence is strong；According to required time and temporally Order is put into developer solution, among fixing solution, and the time is respectively 1~2min, 1min, takes out film, observes band；Exposure is managed The film mark thought is good, indicates sample ID, date, positive and negative, is scanned, maps；As shown in figure iD.

Immune group chemical experiment

1) Paraffin tissue block makes：Take fresh 1~3mm of breast cancer tissue samples³, it is solid with 4% paraformaldehyde (PFA) Determine 24h；Wipe away after the paraformaldehyde on surface with following gradient concentration ethanol dehydration, transparent and waxdip, comprise the following steps that table 1.

Table 1

2) paraffin section makes

It will be fixed on after the wax stone precooling wrapped on slicer, be cut into 5 μm of thin slices, opened up in the Water Tank with Temp.-controlled of exhibition piece instrument After opening, picked up with slide, in 60 DEG C of roasting piece 2h on roasting piece machine.

3) dewaxing, aquation：60 DEG C of dimethylbenzene is handled 2 times, each 20min；Absolute ethyl alcohol is handled 2 times, each 10min； 80% Ethanol Treatment 2min；70% Ethanol Treatment 2min；Distilled water (DDW) aquation 5min；Finally PBS 3 times, every time 3min。

4) cell-permeant, closing endogenous peroxydase：(room temperature, kept away with penetrating liquid infiltration section 30min is closed Light)；PBS solution is washed 3 times, each 3min.

5) antigen retrieval exposure antigenic determinant：Paraffin section is put into the beaker equipped with 0.01M sodium citrate buffer solutions After interior, it is put into pressure cooker and boils 10~15min (to add water in pot), close power supply after boiling, it is down to room temperature naturally.

6) nonspecific proteins is closed：PBS solution is washed 3 times, each 5min；Moisture around is blotted with filter paper, changes pen with group Tissue is enclosed, is then put into after circle inner tissue instills 5%BSA in wet box, room temperature 1h.

7) it is incubated primary antibody：The BSA in section is blotted, is dried with filter paper and serum is remained around tissue, be directly added into primary antibody (1：250) after, it is put into wet box and then 4 DEG C is stayed overnight, second day, being taken out from refrigerator needed 37 DEG C of rewarming 45min.

8) secondary antibody is incubated：Washed 5 times with PBS, each 5min；The water around circle is sucked with filter paper, adds secondary antibody rear chamber Temperature is incubated 1h；Washed 5 times with PBS, each 10min.

9) SP reacts：30min is incubated after adding SP；Washed 5 times with PBS, each 10min.

10) develop the color：Add DAB (quick instill, observe staining conditions), developing time control is in 1~5min, micro- Microscopic observation color change.

11) redye, be dehydrated, be transparent, mounting：With PBS 3 times, each 3min, 5min is rinsed with distilled water afterwards；Add one Haematoxylin dye liquor is dripped, dyes 30s, then running water rinses, then returns blue 5min with PBS；Serial dehydration, step are as follows：50% second Alcohol 10min, 70% ethanol 10min, 95% ethanol 10min, 95% ethanol 10min, absolute ethyl alcohol 10min, absolute ethyl alcohol 10min；It is transparent：Dimethylbenzene handles 10min, and dimethylbenzene is handled 2 times, each 10min；Mounting：Neutral gum；Under the microscope Observe, take pictures；As shown in Fig. 1 E.

As shown in Figure 1A~1E, relative to normal galactophore tissue, breast cancer tissue (triple negative breast cancer tissue and non-three Negative breast cancerous tissue) in SH3BGRL genes equal a large amount expression (including transcription and translation), the SH3BGRL of a large amount expression can be with As the clinical diagnosis of patient with breast cancer and the label of prognosis.

Effects of the embodiment 2SH3BGRL to signal path

Immunoblot experiment

With reference to western blotting method in embodiment 1, in detection MCF-7 cells and overexpression SH3BGRL MCF-7 cells P-AKT473, AKT and p-ERK, ERK expression, as a result as shown in Figure 2 A, MCF-7SH3BGRL represents to be overexpressed in figure SH3BGRL MCF-7 cells.

Mouse is subcutaneously tested into knurl

1) material：Cell line, such as breast cancer MDA-MB-231-Luc cells and MCF-7Luc cells (band luciferase mark Note)；Immune deficient mice of the mouse week old at 4~6 weeks, BALB/c nude mices and NOD/SCID mouse；1ml syringes and 25 × 5/8 syringe needle；Anesthetic (pentobarbital sodium).

2) method

It is prepared by fluorescein：Dissolving fluorescein prepares 15mg/ml storage mother liquor into PBS solution, by a diameter of 0.2 μ M filter sterilization；The fluorescein dosage of every mouse injection is 150mg/kg.

It is prepared by tumour cell：With trypsin digestion and cell, cell is collected, it is then clear with the physiological saline without serum Wash cell；The density of suspension cell is 1 × 10⁸/ 100 μ l physiological saline, are placed standby at room temperature.

Mouse is injected：First luciferin solution is injected intraperitoneally into Mice Body with 25 × 5/8 syringe needles, after 7~8min, abdomen Chamber injecting narcotic carries out mouse anesthesia；After mouse anesthesia, 100 μ l reference substances and testing sample (are contained 1 × 10 with syringe needle⁶ Tumour cell) to be expelled to mouse groin both sides subcutaneous；After mouse revival, it is put into cage and normally raises.

Observation, image scanning：Taking pictures and counting for tumour is carried out to the mouse for being inoculated with tumour cell；As a result such as Fig. 2 B institutes Show, R-SH3BGRL represents to be overexpressed SH3BGRL in figure.

Co-immunoprecipitation experiment

1) material：Cell line, such as co-express GFP-SH3BGRL and GFP-Her2 293T cells, co-immunoprecipitation reagent Box

2) method

Cell is collected, 4,000rpm centrifugation 7min, abandons supernatant；It is (general that appropriate cell pyrolysis liquid is added into cell precipitation 1×10⁶Individual cell adds 120~150 μ l RIPA cell pyrolysis liquids, and adds protease inhibitors)；Crack on ice, juxtaposition In cracking 1h on shaking table；12,000rpm, 4 DEG C of centrifugation 30min, collect supernatant, are transferred to new EP pipes；Add into cell pyrolysis liquid Enter appropriate proteinA/G (adding 50 μ l proteinA/G in the general cell pyrolysis liquid per 1ml), in 4 DEG C of refrigerators, delay Slowly 1h is shaken up, to remove the albumen with proteinA/G non-specific bindings；13,000rpm centrifugation 5min, collect supernatant, transfer Managed to new EP；According to antibody dilution ratio, SH3BGRL antibody is added into supernatant, was slowly mixed in 4 DEG C of refrigerators Liquid；With 10 μ l/1 × 10⁶The amount of individual cell adds proteinA/G, and 4h is slowly shaken up in 10 DEG C of freezers；4 DEG C, 2,500g from Heart sample 3min, collect precipitation；Add the reverse cleaning precipitations of 1ml PBS 3 times, 2,500g centrifuges 1min every time, abandons supernatant；To The PBS that 50 μ l are added in the precipitation that collection obtains is resuspended, and the 5 × loading buffer for adding 15 μ l are carried out Western blotting are tested.

As shown in Fig. 2A~2C, SH3BGRL and Her2, which exists, to interact, excessive in non-triple negative breast cancer cell The SH3BGRL of expression activates PI3K and AKT paths by Her2, promote non-triple negative breast cancer cell in Mice Body into Knurl.

The patient with breast cancer's survival rate of embodiment 3 and the analysis of time-to-live

Collect the long term follow-up treatment of clinical breast cancer patient, the existence after statistics morbidity, treatment after the regular period Or death condition is to judge treatment effect；Curve map is obtained using kaplan-meier algorithms, as a result as shown in Figure 3.

As shown in figure 3, low expression SH3BGRL patient with breast cancer generally has higher survival rate and time-to-live；Will Fig. 2 and Fig. 3 are combined, it can be deduced that the signal transduction pathway of interference SH3BGRL mediations, can be treated the high expression of SH3BGRL and be caused Breast cancer, such as：The antibody for suppressing the siRNA of SH3BGRL mRNA translations or being specifically bound with SH3BGRL.

The dielectrophoresis of embodiment 4

Sample preparation

1) the general processing step of cell sample：Nutrient solution is suctioned out, with pancreatin digestion or cell scraping harvesting；Add Enter PBS solution, 1500g centrifugation 10min, abandon supernatant, be repeated 3 times；Cell is dispensed into 1.5 ml EP pipes, blots residual PBS；Add lysis buffer (1.5 × 10⁶Individual cell about adds 100 μ l lysates), in shaken at room temperature 1h, make its abundant Dissolving；15,000 turns, 4 DEG C of centrifugation 15min；Draw supernatant and with quantification of protein is carried out, preserved in then dispensing to EP pipes It is standby at -78 DEG C.

2) the general processing step of tissue sample：Grind alms bowl to mill tissue, grind to powdered；Every gram of sample adds 0.5ml and split Liquid is solved, is homogenized using tissue homogenizer；Add 50 μ g/ml RNase and 200 μ g/ml DNase, 15min is placed at 4 DEG C；15, 000 turn, 4 DEG C of centrifugation 20min；Collect supernatant, quantification of protein, rearmounted -78 DEG C of preservations of packing.

First to isoelectric focusing

1) IPG adhesive tape aquation and electrophoresis：With sample dissolving buffer (9M urea, 4%CHAPS, 2%IPG buffer solutions, 40mM DTT, 40mM Tris-base) sample dissolution, protein sample concentration do not exceed 10mg/ml, otherwise can cause egg White matter is gathered or precipitated；Appropriate amount of sample solution is drawn according to the pH scopes, length and follow-up colouring method of adhesive tape, then Addition hydrating fluid supplies cumulative volume, and (when adhesive tape length is 7cm, adhesive tape hydrating fluid volume is 125 μ l；When adhesive tape length is 11cm, Adhesive tape hydrating fluid volume is 200 μ l；If adding sample during aquation, this volume is the final volume added after sample), it is put into mark In pseudotype adhesive tape groove, to ensure that sample well into adhesive tape, not add excessive hydrating fluid；From acidic terminal (mark "+" end) The diaphragm of IPG adhesive tape is peelled off in side, glue surface down, the first tip direction by IPG adhesive tape anode tap towards standard type adhesive tape groove It is put into adhesive tape groove, slowly pushes adhesive tape, and move forward and backward, avoids generating bubble, finally put down IPG adhesive tape flush end (negative electrode), Hydrating fluid is soaked whole adhesive tape, and ensure that the both ends of adhesive tape and the electrode at the both ends of groove contact；Covered in IPG adhesive tape appropriate Immobiline DryStrip covering oil, closes the lid；By the sophisticated backplate of standard type adhesive tape groove and IPGphor instruments Anode platform contact；The flush end backplate of adhesive tape groove contacts with the cathode platform of IPGphor instruments；IPGphor instrument is set Device operational factor：The voltage of IPG adhesive tape aquations, temperature and time；Using 11cm adhesive tape, aquation is arranged to voltage 50V, 12h (17 DEG C), then aquation loading；After loading, operation program is：

Pay attention to：The placed adhesive tape number of selection, the carrying current (50 μ A/ roots) of every adhesive tape is set, isoelectric focusing is set When temperature (17 DEG C).

2) balance of IPG adhesive tape：Take out IPG adhesive tape (7cm adhesive tape) to be respectively put into 15ml BD pipes, shaken on shaker 15min is swung, outwells level pad I；Appropriate level pad II is added, 15min is vibrated on shaker, it is slow to outwell balance Fliud flushing II (IPG adhesive tape equilibrium liquid dosages：When adhesive tape length is 7cm, equilibrium liquid volume is 2.5~5ml/ bars；Adhesive tape length is During 11cm, equilibrium liquid volume is 5~10ml/ bars)；With deionized water rinse IPG adhesive tape 1s, the edge of adhesive tape is placed in filter paper Upper a few minutes, to remove unnecessary level pad；The transfer of IPG adhesive tape：IPG adhesive tape is placed between glass plate On gel face, adhesive tape is set to support film close to one piece of glass plate therein, lightly to push down on IPG adhesive tape with a thin chi, make Whole adhesive tape lower edge and the upper surface of plate glue completely attach to；Ensure between IPG adhesive tape and plate glue and glass plate and modeling Material supports intermembranous bubble-free to produce；Add molecular weight marker proteins matter：Standard protein is added to 15~20 μ l volume IEF loading filter papers, loading filter paper is put on a glass, and a certain amount of protein standard solution is added into loading filter paper On piece, loading filter paper is then placed on IPG adhesive tape end side with tweezers, contacted with the gel surface of plate glue；Finally use Agarose sealing fluid is bound, and is completely covered IPG adhesive tape with a small amount of sealing fluid (about 1~1.5ml), herein mistake Bubble is not produced in journey.

Second to SDS electrophoresis

Respective concentration acrylamide gel needed for preparation；The IPG adhesive tape balanced is immersed in electrode buffer several seconds； By IPG adhesive tape being positioned in SDS glue surfaces carefully, and gently pressure makes IPG adhesive tape fully be combined with SDS glue surfaces, SDS molecular weight marks The agarose solution (75 DEG C) of 2ml heat to be placed on the alkaline end of adhesive tape, will definitely be covered above, agarose is solidified in 5min, Remaining IPG adhesive tape repeats aforesaid operations；Glue box are inserted in electrophoresis tank, start electrophoresis；When Bromophenol Blue dye moves to glue Bottom margin can terminate electrophoresis；The glue run is transferred in colouration box fixed, preparation dyeing.

The detection of protein after dielectrophoresis

1) classical Coomassie brilliant blue dyeing procedure：20% (w/v) trichloroacetic acid fixes 1h；0.1%G-250 (is dissolved in The acetic acid of 40% ethanol/10%) dyeing 2h；Destainer (acetic acid of 40% ethanol+10%) decolourizes 2 times, each 30min；Deionized water Clean 30min；As a result it is as shown in Figure 4.

2) silver staining

Glue is fixed：The acetic acid of 50% methanol+5% fixes 20min；50% methanol washs 10min；Deionized water is washed 10min；Sensitization：0.02% sodium thiosulfate 2min；Deionization is washed 2 times, each 1min；Dyeing：Glue, which immerses, contains 0.08% first The 0.1%AgNO of aldehyde₃In solution, lucifuge is incubated 20min；Deionized water is washed 2 times, each 5s；Develop the color 1~10min；Terminate： 5% acetic acid solution reacts 10min, deionized water cleaning 5min；As a result it is as shown in Figure 5.

As shown in figure 4, for normal galactophore tissue, what the SH3BGRL in breast cancer tissue's tumour cell was combined Albumen changes；As shown in figure 5, for normal galactophore tissue, the SH3BGRL in breast cancer tissue's tumour cell The isoelectric point and molecular weight of itself change.SH3BGRL is proved to be tumor suppressor gene in other tumour cells, but A large amount is expressed in breast cancer tissue's tumour cell, implies the SH3BGRL accumulated in breast cancer tissue's tumour cell and has The SH3BGRL of function of tumor inhibition is simultaneously differed, and the reason of SH3BGRL in breast cancer tissue's tumour cell is also confirmed that by experiment Change property to change, form different protein complexes, lose tumor suppression person's character and be converted into the tumor promotion factor.Cause This, SH3BGRL physicochemical property can also turn into as the label of breast cancer clinical diagnosis and prognosis, and SH3BGRL The target spot of breast cancer treatment.

Finally, it should be noted that above example to illustrate technical scheme rather than to the present invention protect The limitation of scope is protected, although being explained in detail with reference to preferred embodiment to the present invention, one of ordinary skill in the art should Understand, technical scheme can be modified or replaced on an equal basis, without departing from the essence of technical solution of the present invention And scope.

Claims (8)

1. Applications of the 1.SH3BGRL or SH3BGRL mRNA in screening or preparing the kit of breast cancer clinical diagnosis or prognosis.
2. 2. SH3BGRL or SH3BGRL mRNA reagent is detected in the kit of breast cancer clinical diagnosis or prognosis is prepared Using.
3. The application of 3.SH3BGRL genes, SH3BGRL mRNA or SH3BGRL in screening or preparing the medicine for the treatment of breast cancer.
4. Application of the 4.SH3BGRL inhibitor in the medicine for preparing treatment breast cancer.
5. 5. application according to claim 4, it is characterised in that the SH3BGRL inhibitor includes suppressing SH3BGRL The siRNA of mRNA translations or the antibody specifically bound with SH3BGRL.
6. 6. a kind of breast cancer clinical diagnosis or the kit of prognosis, it is characterised in that：The kit includes measure SH3BGRL's The specific primer of mRNA transcription amounts, determine the antibody of SH3BGRL translation amounts or detect the reagent of SH3BGRL physicochemical properties.
7. A kind of 7. medicine for treating breast cancer, it is characterised in that：Including SH3BGRL inhibitor.
8. 8. medicine as claimed in claim 7, it is characterised in that：The SH3BGRL inhibitor includes the mRNA for suppressing SH3BGRL The siRNA of translation or the antibody specifically bound with SH3BGRL.