CN107014998B - A kind of lung cancer regulatory factor SRSF5 and its inhibitor and application - Google Patents
A kind of lung cancer regulatory factor SRSF5 and its inhibitor and application Download PDFInfo
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Abstract
The invention discloses a kind of lung cancer regulatory factor SRSF5 and its inhibitor and applications.The present invention plays a driving role for lung cancer by the regulation unbalance shaft that the tumor formation in nude mice of the proliferation experiment of cytology level, plate clone formation experiment and Ex vivo animal integral level demonstrates SRSF5-CCAR1.Above-mentioned description of test SRSF5-CCAR1 signal shaft can be used as potential lung cancer therapy target, enrich the understanding to the activity regulation mechanism of splicing factor.The screening of micromolecular inhibitor or montage Active Regulation inhibitor based on SRSF5-CCAR1 signal shaft will provide new thinking and important scientific basic for the targeted therapy of lung cancer.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of lung cancer regulatory factor SRSF5 and its inhibitor and application.
Background technique
There are alternative splicing, alternative splicing determines the diversity of RNA and to protein 95% gene in eucaryote
Expression pattern have an important influence on.The dynamic regulation network of alternative splicing is by several cis-acting elements and trans-acting factor
Composition: usual arginine serine enrichment splicing factor (SRSF) is incorporated in explicit leaming enhancer and introne montage enhancing
On son, and dyskaryosis ribonucleoprotein (hnRNP) is incorporated on explicit leaming repressor and introne montage repressor, the two
There are the relationships of vying each other on room and time.Classical montage activation is by SR protein binding in corresponding cis-splicing
Constitutive protein U1, U2 and its correlation confactor of montage corpusculum are recruited on element, and then the exon of alternative splicing is discharged；
And in montage process of inhibition, dyskaryosis ribonucleoprotein cuts off the exon of alternative splicing after forming lasso structure.Cause
This, the activity of splicing factor and the finely regulating of content are particularly important for the assembling of montage corpusculum and Function.
SRSF points are enriched with this type montage for classic arginine serine concentration type splicing factor and arginine serine
Factor two major classes, classic SR albumen have 12 members, and the structural domain of conservative is usually the 1-2 RNA knowledge for being located at N-terminal
Other structural domain and arginine serine rich region positioned at C-terminal, the latter are also generally considered the posttranslational modification of SR albumen
The high-frequency region of generation.Current more acetylations and ubiquitination group data set are shown: SRSF5 is only one while depositing
In two kinds of posttranslational modifications and SR type splicing factor (decorating site K125) that decorating site height is overlapped, therefore its functional character
It is worth inquiring into adjustment mechanism.The alternative splicing of Abnormal regulation is the classical mark of tumour cell.However, crucial in alternative splicing
The Active Regulation of ingredient splicing factor is known little about it.
CCAR1 widely participates in the adjusting of cell cycle and Apoptosis and by notch signal stimulus, but so far,
The montage Active Regulation relationship of CCAR1 is known little about it.Shearing substrate of the CCAR1 as SRSF5, shearing product share long and short two kind
Form A CAR1L and CCAR1S, CCAR1L saves complete transcript and CCAR1S is that the 15-22 exon of CCAR1 occurs
Product after alternative splicing.
Summary of the invention
It is an object of the present invention to provide the new applications for inhibiting SRSF5 expression and/or active substance.
The present invention provides inhibit SRSF5 expression and/or active substance to have at least one in following (1)-(3) in preparation
Application in the product of kind function:
(1) treatment and/or pre- preventing tumor；
(2) inhibit the growth of tumour cell；
(3) CCAR1 alternative splicing product assay in modulate tumor cell.
In above-mentioned application, the growth for inhibiting tumour cell is embodied in the weight for reducing tumour cell and/or reduces swollen
The volume of oncocyte and/or the ratio for improving apoptotic tumor cell in situ.
In above-mentioned application, CCAR1 alternative splicing product assay is embodied in raising tumour cell in the modulate tumor cell
CCAR1 short-form variable sheer product contains in the content and/or reduction tumour cell of middle CCAR1 long form variable sheer product
Amount and/or the ratio for improving CCAR1 long form variable sheer product and CCAR1 short-form variable sheer product in tumour cell.
In above-mentioned application, the inhibition SRSF5 expression and/or active substance are anti-SRSF5 antibody, SRSF5 small molecule
The RNA molecule of inhibitor, SRSF5 soluble protein or interference SRSF5 expression.
In above-mentioned application, the RNA molecule of the interference SRSF5 expression is to interfere the shRNA of SRSF5 expression.
In above-mentioned application, the shRNA of the interference SRSF5 expression is RNA molecule shown in sequence 1 or sequence 2.
It is a further object to provide a kind of products.
The active constituent of product provided by the invention is to inhibit SRSF5 expression and/or active substance, the use of the product
Way is at least one of following (1)-(3):
(1) treatment and/or pre- preventing tumor；
(2) inhibit the growth of tumour cell；
(3) CCAR1 alternative splicing product assay in modulate tumor cell.
In the said goods, the growth for inhibiting tumour cell is embodied in the weight for reducing tumour cell and/or reduces swollen
The volume of oncocyte and/or the ratio for improving apoptotic tumor cell in situ.
In the said goods, CCAR1 alternative splicing product assay is embodied in raising tumour cell in the modulate tumor cell
CCAR1 short-form variable sheer product contains in the content and/or reduction tumour cell of middle CCAR1 long form variable sheer product
Amount and/or the ratio for improving CCAR1 long form variable sheer product and CCAR1 short-form variable sheer product in tumour cell.
In the said goods, the inhibition SRSF5 expression and/or active substance are anti-SRSF5 antibody, SRSF5 small molecule
The RNA molecule of inhibitor, SRSF5 soluble protein or interference SRSF5 expression.
In the said goods, the RNA molecule of the interference SRSF5 expression is to interfere the shRNA of SRSF5 expression.
In the said goods, the shRNA of the interference SRSF5 expression is RNA molecule shown in sequence 1 or sequence 2.
In above-mentioned application or product, the tumour is non-small cell lung cancer, and the tumour cell is that non-small cell lung cancer is thin
Born of the same parents, the non-small cell lung cancer cell are specially A549 cell.
Final object of the present invention is to provide following 1) -3) in any new application as target spot:
1)SRSF5；
2) SRSF5-CCAR1 signal shaft；
3) CCAR1 alternative splicing product.
In above-mentioned application, the SRSF5-CCAR1 signal shaft refers to that the expression of SRSF5 and/or activity decline, CCAR1 are short
The ratio of form alternative splicing product (CCAR1S) declines, i.e. SRSF5 expression and CCAR1 short-form alternative splicing product
(CCAR1S) ratio is positively correlated.
In above-mentioned application, the CCAR1 alternative splicing product be CCAR1 short-form alternative splicing product (CCAR1S) and/
Or CCAR1 long form alternative splicing product (CCAR1L).
The present invention provides above-mentioned 1) -3) in it is any as target spot in exploitation treatment and/or prevention non-small cell lung cancer
Drug in application.
The present invention is by carrying out western blot and immunohistochemical analysis discovery to 60 non-small cell lung cancer clinical samples:
In the clinical sample of non-small cell lung cancer, the protein level of SRSF5 and the protein level of acetylation SRSF5 are raised, and are presented
Dominant positive correlation, at the same the protein level of the protein level of SRSF5 and acetylation SRSF5 with CCAR1 short-form alternative splicing
Ratio have dominant positive association；Then experiment is formed by the proliferation experiment of cytology level, plate clone and moved in vitro
The regulation unbalance shaft that the tumor formation in nude mice of object integral level demonstrates SRSF5-CCAR1 promotes to make for the generation of lung cancer
With.Above-mentioned description of test SRSF5-CCAR1 signal shaft can be used as potential lung cancer therapy target, enrich to splicing factor
The understanding of activity regulation mechanism.Micromolecular inhibitor or montage Active Regulation inhibitor based on SRSF5-CCAR1 signal shaft
Screening will provide new thinking and important scientific basic for the targeted therapy of lung cancer.
Detailed description of the invention
Fig. 1 is the protein level testing result of SRSF5 in non-small cell lung cancer and neighbouring cancer beside organism.
Fig. 2 be non-small cell lung cancer and its in cancer beside organism, the immunohistochemistry of SRSF5 and acetylation SRSF5 detects
Result figure.
Fig. 3 is the protein level testing result of acetylation SRSF5 in non-small cell lung cancer and neighbouring cancer beside organism.
Fig. 4 be non-small cell lung cancer and its in cancer beside organism, the ratio of two kinds of transcripts of CCAR1L and CCAR1S is examined
Mapping.
Fig. 5 is CCAR1S/CCAR1L relative scale in the non-small cell lung cancer matched for 60 and neighbouring cancer beside organism
Results of statistical analysis.
Fig. 6 is the testing result of the expression of SRSF5 after striking low SRSF5.
Fig. 7 is the testing result of CCAR1S/CCAR1L ratio variation after striking low SRSF5.
Fig. 8 is the testing result of tumour growth volume after striking low SRSF5 in Lines A549.
Fig. 9 is the testing result of tumor weight after striking low SRSF5.
Figure 10 is the primary tumor TUNEL dyeing testing result after striking low SRSF5.
Figure 11 is the detection of the tumor growth rate in striking low SRSF5 and the redemption experiment for being overexpressed CCAR1L and CCAR1S
As a result.
Figure 12 is tumour growth volume and weight in the redemption experiment for strike low SRSF5 and overexpression CCAR1L and CCAR1S
Testing result.
Figure 13 is to save primary tumor TUNEL in experiment after striking low CCAR1S to dye testing result.
Specific embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Quantitative test in following embodiments, is respectively provided with three repeated experiments, and results are averaged.
The detection of the protein level and its Acetylation Level of SRSF5 in embodiment 1, non-small cell lung cancer clinical sample
One, the detection of the protein level of SRSF5
1, immunoblot experiment
(1) collection of sample
(every sample includes the cancerous tissue and its phase of non-small cell lung cancer to the non-small cell lung cancer tumor sample of 60 pairings
Adjacent normal tissue) collected from PLA General Hospital department of general surgery, all clinical samples all have necessary clinical information and acquisition
Informed consent, the acquisition and storage of sample meet clinical criteria and regulation.
(2) Western Blot is detected
After sample is collected, sample tissue is ground using liquid nitrogen method, is ground into fine-powder, according to every 0.1g/100
The dissolution of RIPA lysate is added in μ l, and sample detection after isometric sample-loading buffer boiling water bath 5-10min is added.Concrete operations
Step: glue 90V is concentrated in SDS-PAGE electrophoresis, and 20-30min will be electric after albumen pre-dyed Marker is completely separable in separation gel
Pressure is adjusted to 180V；After bromophenol blue is completely separable, albumen electricity is gone on NC film；By NC film transfer to containing 5% skim milk
TBST in close 1h；Primary antibody is added after closing: 4 DEG C of SRSF5 (abcam, ab67175 1:400) overnight or room temperature 3h；TBST
Film 3 times are washed, each 10min；It is incubated at room temperature secondary antibody: sheep anti mouse secondary antibody (Jackson ImmunoResearch, 115-035-003)
Or goat-anti is washed 3 times in rabbit secondary antibody (Jackson ImmunoResearch, 111-035-003) with TBST, and ECL chemistry bottom is added
Object luminescence reagent darkroom exposure development；Usually retain short exposure (within 30s), middle exposure (5-10min) and length expose (3 hours
More than) different exposure stage films it is several, hypersensitivity kit can be added when necessary.
Western Blot testing result is as shown in Figure 1, as can be seen from the figure: the protein level of SRSF5 is non-small thin
There are significant difference in born of the same parents' lung cancer and its cancer beside organism, the protein expression level of SRSF5 is apparently higher than correspondence in cancerous tissue (T)
Cancer beside organism (N) expression.
2, immunohistochemical experiment
Non-small cell lung cancer paraffin specimen is made using Lung Cancer Tissue Microarray (being purchased from Shanghai core excusing from death object Co., Ltd)
At organization chip, organization chip is detected using Immunohistochemical Method, carries out staining analysis using the corresponding tissue samples of serial section,
The antibody and concentration that the immunohistochemistry of tumour chip uses are as follows: SRSF5 (1:100, Novus Bio).Specific step is as follows:
1) white tiles dewaxes: each 10min of dimethylbenzene I, II；
2) be sliced rehydration: the alcohol time of 100%I, 100%II, 95%, 90%, 80%, 70% be followed successively by 8min,
8min,6min,5min,3min,3min；
3) distilled water washs 2min × 3；
4) 3% hydrogen peroxide removes Endogenous peroxidase 15min, is protected from light incubation；
5)PBS 2min×3；
6) antigen retrieval: citrate reparation liquid is boiled used time 10min or so by high fire in micro-wave oven, slowly cools to room
Temperature；
7) PBS is washed 3 times, each 3min；
8) it stays overnight for 37 DEG C of lowlenthal serum 1h or 4 DEG C of closing；
9) liquid is gently got rid of, drip primary antibody for hanging side, 4 DEG C of overnight incubations；
10) plus secondary antibody adjuvant, 37 DEG C, 30min；
11)PBS 3min×3；
12) plus horseradish peroxidase-labeled secondary antibody, 37 DEG C, 30min；
13)PBS 3min×3；
14) DAB develops the color, and under the microscope, A:B:C 6:6:6/500 μ l keeps chromogenic reaction uniform；
15) distilled water flushing, bush uniformly dyeing core 10-30s, tap water rinse 2min, and alcohol hydrochloric acid breaks up 23s, tap water
It rinses；
16) it is dehydrated: 70%, 80%, 90%, 95%, 100% each 2min；
17) transparent: dimethylbenzene I, II distinguish 5min；
18) resinene mounting under the microscope, and acquires photo using Nikon imaging system.
Shown in testing result such as Fig. 2 (the first row), IHC coloration result shows: in non-small cell lung cancer, SRSF5 is thin
The coloring of karyon significantly increases, and illustrates that the protein expression level of SRSF5 in non-small cell lung cancer significantly raises.
Two, the detection of SRSF5 Acetylation Level
1, immunoblot experiment
(1) collection of sample
With in step 11 (1).
(2) Western Blot is detected
Detection method in step 11 (2), used in antibody be Ac-SRSF5 (the limited public affairs of the farsighted star in Shanghai biology
Department's self-control, 1:300).
Testing result as shown in figure 3, the protein level of acetylation SRSF5 (Ac-SRSF5) in non-small cell lung cancer and its cancer
There are significant difference in the tissue of side, the protein expression level of Ac-SRSF5 is apparently higher than group by corresponding cancer in cancerous tissue (T)
Knit the expression of (N).
2, immunohistochemical experiment
With in step 12 experimental method.Antibody concentration used in detection is that (the farsighted star biology in Shanghai is limited by Ac-K125
Company's self-control, 1:50).
Shown in testing result such as Fig. 2 (the second row), IHC coloration result shows: in non-small cell lung cancer, acetylation
SRSF5 (Ac-SRSF5) is significantly increased in the coloring of nucleus, illustrates the acetylation SRSF5 (Ac- in non-small cell lung cancer
SRSF5 protein expression level) significantly raises.SRSF5 protein level and acetylation SRSF5 protein level are positively correlated.
Three, the ratio detection of CCAR1 variable sheer product
Shearing substrate of the CCAR1 as SRSF5, shearing product share long and short two kind form A CAR1L and CCAR1S, SRSF5
Protein expression level and the protein expression level of acetylation SRSF5 influence the ratio of CCAR1 variable sheer product, the step
Using the cDNA of non-small cell lung cancer tumor tissues sample as template, carried out using the primer that specificity distinguishes long and short two kind form real
When quantitative PCR, detect non-small cell lung cancer tumor tissues sample in long and short two kind form relative abundance and content.Wherein, it examines
The primer sequence for surveying long form variable sheer product (CCAR1L) is 5 '-GTTTCAATCCCGCCAACGCA-3 ' and 5 '-
CCGAACACGTCTCCTGCTC-3'；The primer sequence for detecting short-form variable sheer product (CCAR1S) is 5 '-
ACTCCTTGTAGCGACCTGG-3 ' and 5 '-CCACGCTATGATCAGAGCTACG-3 '.Specific step is as follows:
1) homogenization is handled: extracting RNA from the tumor sample tissue grinder of pairing using grinding rod, 1mlTrizol is added；
2) it mutually separates: homogenization sample 5min at room temperature；
3) RNA is extracted: 0.2ml/ chloroform is added: 1mlTrizol reagent carefully covers EP pipe lid, violent whipping centrifuge tube
15-20s makes its mixing, incubation at room temperature 2-3min；4 DEG C, 12000rpm, it is centrifuged 15min.Mixture is divided into three layers after centrifugation: most
Bottom red-phenol chloroform, intermediate and upper strata aqueous phase, RNA are located at water phase, water phase about of the total volume 60%；
4) it precipitates RNA: water phase is transferred to new centrifuge tube；0.5ml isopropanol/1mlTrizol is added, makes RNA precipitate；
Incubation at room temperature 10min, 4 DEG C, 12000rpm is centrifuged 10min, and incubation reacts water phase sufficiently with reagent.
5) it washs RNA: removing supernatant, be added 75% ethyl alcohol of 1ml/1ml Trizol, vortex sample, 4 DEG C, 7000rpm,
5min。
6) it re-dissolves: after RNA slightly dries, exposing whiteness, unsuitable too dry reduces the dissolution of RNA.It is added
20-30 μ l DEPC water, 55-60 DEG C of water-bath 10min are all mixed after dissolution with pipette tips.
7) RNA quality testing: gel electrophoresis result be shown as three bands or A260/A280 ratio be 1.8, RN content=
A260 × extension rate.
8) synthesis of the first chain uses the kit of TOYOBO company, and reaction system is as follows: 2 × mix 5ul+RNA (total amount
1 μ g or less)；Response procedures: 37 DEG C of 20min, 95 DEG C of 5min, 4 DEG C of preservations.
Testing result is as shown in Figure 4 and Figure 5, as can be seen from the figure: in non-small cell lung cancer sample, CCAR1S/
CCAR1L ratio is significantly increased compared with Normal group, is shown for the statistical analysis of 60 samples: the result has significant
Property.
In summary: in the clinical sample of non-small cell lung cancer, the protein level of SRSF5 and the egg of acetylation SRSF5
The average significant up-regulation of plain boiled water, while the protein expression level of key component splicing factor SRSF5 and acetylation in alternative splicing
Ratio of the protein level of SRSF5 with CCAR1 short-form alternative splicing product (CCAR1S) is positively correlated.
Embodiment 2 inhibits application of the substance of SRSF5 expression in treatment non-small cell lung cancer
One, the building of low SRSF5 cell and its related revolution cell strain is struck
1, the building and identification of low SRSF5 cell are struck
(1) expression plasmid of low SRSF5 is struck
By sh-SRSF5-1 be inserted into pLKO.1puro slow virus carrier (carrier be purchased from addgene, #8453) AgeI and
Between the site EcoRI, the expression plasmid #1 for striking low SRSF5 is obtained；
By sh-SRSF5-2 be inserted into pLKO.1puro slow virus carrier (carrier be purchased from addgene, #8453) AgeI and
Between the site EcoRI, the expression plasmid #2 for striking low SRSF5 is obtained；
By between the site AgeI and EcoRI of control sequence shRNA insertion pLKO.1puro slow virus carrier, control matter is obtained
Grain.ShRNA sequence is as follows:
Sh-SRSF5-1:GAGAAGGACGTGGAAAGATT (sequence 1)；
Sh-SRSF5-2:GACGGATAAGAGATATTGATCT (sequence 2)；
Control (control sequence shRNA): TTCTCCGAACGTGTCACGTAT.
(2) slow virus packaging and purifying
By the expression plasmid #1 for striking low SRSF5 constructed in step (1), strikes the expression plasmid #2 of low SRSF5 and compare matter
Grain carries out slow virus packaging and purifying, and formation is coated with the expression plasmid #1 for striking low SRSF5, the expression plasmid #2 for striking low SRSF5
With the viral concentration liquid of control plasmid.Specific step is as follows:
1) 1:3 is passed on 2 times after 293T cell (Shanghai Bo Gu biotech firm, CBP60439) recovery, and then paving to 60mm is trained
Support ware；
2) liquid is changed after 18 hours, is transfected after maintaining optimal cell growth state 6-8 hours, and transfection specific steps are such as
Under:
To strike the expression plasmid #1 of low SRSF5, packaging plasmid psPAX2 (biovector, BioVector1058 1-52,
Similarly hereinafter) and pMD2.G (BioVector1058 1-53, similarly hereinafter) according to mass ratio be 1:3:4 ratio import 293T cell jointly
In；
The ratio that expression plasmid #2, the packaging plasmid psPAX2 and pMD2.G of low SRSF5 are 1:3:4 according to mass ratio will be struck
It is common to import in 293T cell；
Control plasmid, packaging plasmid psPAX2 and pMD2.G are imported into 293T according to the ratio that mass ratio is 1:3:4 jointly
In cell.
3) liquid complete medium is changed after 12h: then collecting viral supernatants after 24,48,72 hours；
4) it after collecting neat supernatant, is filtered with 0.45 μm of aperture sterile filters；Viral supernatants are concentrated using super filter tube
The ratio that (Lenti-Concentin Virus Precipitation Solution, Excell Bio) is 4:1 according to volume ratio
Example mixes, 4 DEG C of standing at least 12h, with refrigerated centrifuge 3000g, 30min, 4 DEG C；
5) supernatant is abandoned, precipitating is collected；(concentration ratio for being 1:100 according to volume ratio) is resuspended to precipitating with PBS,
It obtains being coated with the expression plasmid #1 for striking low SRSF5, strike the expression plasmid #2 of low SRSF5 and the viral concentration liquid of control plasmid.-
80 DEG C of refrigerators save.
(3) aim cell is infected
The viral concentration liquid inductance for being coated with the expression plasmid #1, #2 and control plasmid that strike low SRSF5 dye is used into F12K respectively
The aim cell A549 (being purchased from ATCC, article No. ATCC CRM-CCL-185) of culture medium (cellgro, 10-025-CV) culture, sense
Dye ratio controls between 1:2-1:3, is screened one week using 2 μ g/ml puromycin, obtains the stabilization cell for striking low SRSF5
It is #1, the stable cell lines #2 and control cell for striking low SRSF5.
(4) identification of low SRSF5 cell is struck
Using Western Blot detection strike low SRSF5 stable cell lines #1, strike low SRSF5 stable cell lines #2 and
The protein expression level of the CCAR1L and CCAR1S of the SRSF5 protein expression level and long and short two kind form of control cell.
Protein expression level testing result such as Fig. 6 of SRSF5.It can be seen from the figure that striking the stabilization cell of low SRSF5
It is #1 and the stable cell lines #2 successful knockout SRSF5 for striking low SRSF5.
The expression testing result of the CCAR1L and CCAR1S of long and short two kind form are as shown in Figure 7.It can from figure
Out, CCAR1 long form variable sheer product in the stable cell lines #1 for the striking low SRSF5 and stable cell lines #2 for striking low SRSF5
(CCAR1L) ratio significantly improves.Illustrate SRSF5 protein level and CCAR1 short-form alternative splicing product (CCAR1S) at just
It is related.
2, the building and identification of related revolution cell strain after low SRSF5 cell are struck
(1) it strikes low SRSF5 and turns round the building of CCAR1L cell strain
1) building of expression plasmid
Sh-SRSF5-1 shown in sequence 1 is inserted into pMKO.1puro retroviral vector, and (carrier is purchased from addgene, #
8452) between the site AgeI and EcoRI, the expression plasmid for striking low SRSF5 is obtained；
By between the site AgeI and EcoRI of control sequence shRNA insertion pMKO.1puro slow virus carrier, control matter is obtained
Grain；
By the insertion of CCAR1L full length sequence shown in sequence 3 pQCXIH hygro retrovirus, (carrier is purchased from
Addgene, #33091) the site NotI and XhoI between, obtain stablize be overexpressed CCAR1L expression plasmid.
2) packaging and purifying of retrovirus
The expression plasmid for striking low SRSF5, control plasmid and the expression matter for stablizing overexpression CCAR1L that step 1) is obtained
Grain carries out retrovirus packaging and purifying, respectively obtains and is coated with the expression plasmid for striking low SRSF5, control plasmid and stablized
Express the viral concentration liquid of CCAR1L expression plasmid.Specific method is with 1 in step 1 (2)).
3) aim cell is infected
In view of the amphicheirality of selection markers, first respectively with being coated with the expression plasmid, control plasmid that strike low SRSF5
Viral concentration liquid inductance contaminates A549 cell (infection proportion is adjusted to 1:3).After 48 hours, the first run screening for striking low cell strain is carried out
(3ug/ml puromycin, 1 week).The viral concentration liquid inductance for stablizing the expression plasmid for being overexpressed CCAR1L will be then coated with to contaminate
The positive cell screened to the first run (infection proportion is adjusted to 1:2).It is after 36 hours, the positive for being coated with CCAR1L (redemption) is thin
Born of the same parents carry out two wheel screenings (hygromycin, 350mg/ml, 2 week), finally obtain and strike low SRSF5 and turn round CCAR1L cell strain.
Polybrene (8ug/ml) can be added in virus infection and improve efficiency of infection.
(2) it strikes low SRSF5 and turns round the building of CCAR1S cell strain
By NotI and XhoI of the insertion pQCXIH hygro retroviral vector of CCAR1S full length sequence shown in sequence 4
Between point, obtain stablizing the expression plasmid for being overexpressed CCAR1S.
The expression plasmid that stablizing in above-mentioned steps (1) is overexpressed CCAR1L is replaced with and stablizes the table for being overexpressed CCAR1S
It up to plasmid, and keeps remaining step constant, obtain striking low SRSF5 and turns round CCAR1S cell strain.
Two, tumor formation in nude mice
By test, with Balb/c nude mice (male, 5-6 week old size, 18.0 ± 2.0g), raising is in SPF grades of animal houses, to small
After mouse adapts to SPF environment, it is divided into two groups using the principle being grouped at random, following each group is divided into according to processing mode difference:
1, control group
The control cell (3 × 10 that will be prepared in the 1 of step 16/ ml) and Matrigel (BD Bioscience, #
356234) it is mixed according to the ratio that volume ratio is 0.25:1, obtains control systems；And by 100ul control systems subcutaneous injection in
At the armpit of nude mice.
2, low SRSF5 group is struck
The stable cell lines #1 (3 × 10 for striking low SRSF5 that will be prepared in the 1 of step 16/ ml) and Matrigel according to body
Product is mixed than the ratio for being 0.25:1, obtains striking low SRSF5 system；And 100ul is struck into low SRSF5 system subcutaneous injection in nude mice
Armpit at.
3, it strikes low SRSF5 group and turns round CCAR1L group
It strikes low SRSF5 by what is prepared in the 2 of step 1 and turns round CCAR1L cell strain (3 × 106/ ml) and Matrigel press
It is mixed according to the ratio that volume ratio is 0.25:1, obtains striking low SRSF5 and turn round CCAR1L system；And 100ul is struck into low SRSF5 simultaneously
The subcutaneous injection of CCAR1L system is turned round at the armpit of nude mice.
4, it strikes low SRSF5 group and turns round CCAR1S group
It strikes low SRSF5 by what is prepared in the 2 of step 1 and turns round CCAR1S cell strain (3 × 106/ ml) and Matrigel press
It is mixed according to the ratio that volume ratio is 0.25:1, obtains striking low SRSF5 and turn round CCAR1S system；And 100ul is struck into low SRSF5 simultaneously
The subcutaneous injection of CCAR1S system is turned round at the armpit of nude mice.
There is tumour in 7-10 days control groups after injection, are measured twice a week to tumor size using vernier caliper, swell
The size calculation method of tumor follows following formula L × W2× 0.52 (L represents maximum gauge, and W represents minimum diameter).35-40 days
Afterwards, nude mice anesthesia is put to death, and is then cut out solid tumor and is weighed and recorded, and is fixed using paraformaldehyde, subsequent HE dye
Color determines the tissue morphology of tumour to distinguish cancer and cancer beside organism.After tumor tissues fix 16 hours by formalin, according to
Classical paraffin imbedding is handled and is carried out paraffin section, tests detection tumour cell original position apoptosis feelings followed by TUNEL
Condition, related experimental methods are referring to Promega DeadEndTMColorimetric TUNEL System kit specification.It is above-mentioned
Paraffin section step:
1) be put into embedded box after the tumor tissues fixed being repaired block, according to 70%, 80%, 90%I, 90%II,
95%I, 95%II, 100%I, 100%II serial dehydration, time are respectively 10min, 10min, 8min, 8min, 5min,
5min, 3min, 3min；
2) the transparence tissue in dimethylbenzene: each 10min of dimethylbenzene I, II；
3) waxdip: wax I, each 20min of wax II；
4) it is embedded using Leica paraffin wax embedding, the step of according to wax-tissue-wax；
5) it is sliced according to 3.5 μ m thicks；
6) by exhibition piece machine flattening and dry piece machine on copy piece 6 hours or more, long-term preservation can be placed in 4 DEG C, using it is preceding
60 DEG C of drying overnight.
Gross tumor volume shows with the testing result of weight: comparing with control group, strikes the volume of the nude mouse tumor of low SRSF5 group
It is obviously reduced (Fig. 8), weight is substantially reduced (Fig. 9).TUNEL laboratory test results show: tumour cell original position apoptosis ratio is bright
It is aobvious to increase (Figure 10).Further, strike low SRSF5 horizontal base upper rotary CCAR1L and CCAR1S discovery, and if only if return
After turning CCAR1S, nude mice tumor formation volume becomes larger (Figure 11), and weight increases (Figure 12), and tumour cell original position apoptosis ratio reduces (figure
13).Illustrate that SRSF5 albumen is grown by CCAR1 alternative splicing product assay modulate tumor cell in regulating cell.
Sequence table
<110>INST OF EMISSION & RADIATION M
<120>a kind of lung cancer regulatory factor SRSF5 and its inhibitor and application
<160>4
<210>1
<211>20bp
<212>DNA
<213>artificial sequence
<220>
<223>
<400>1
gagaaggacg tggaaagatt 20
<210>2
<211>22bp
<212>DNA
<213>artificial sequence
<220>
<223>
<400>2
gacggataag agatattgat ct 22
<210>3
<211>3453bp
<212>DNA
<213>artificial sequence
<220>
<223>
<400>3
atggctcaat ttggaggaca gaagaatccg ccatgggcta ctcagtttac agccactgca 60
gtatcacagc cagctgcact gggtgttcaa cagccatcac tccttggagc atctcctacc 120
atttatacac agcaaactgc attggcagca gcaggcctta ccacacaaac tccagcaaac 180
tatcagttaa cacaaactgc tgcattgcag caacaagccg cagctgcagc agctgcatta 240
caacagcaat attcacaacc tcagcaggcc ctgtatagtg tgcaacaaca gttacagcaa 300
ccccagcaaa ccctcttaac acagccagct gttgcactgc ctacaagcct tagcctgtct 360
actcctcagc caacagcaca aataactgta tcatatccaa caccaaggtc cagtcaacag 420
caaacccagc ctcagaagca gcgtgttttc acaggggtgg ttacaaaact acatgatacg 480
tttggatttg tggatgaaga tgtattcttt cagcttagtg ctgtcaaagg gaaaaccccc 540
caagtaggtg acagagtatt ggttgaagct acttataatc ctaatatgcc ttttaaatgg 600
aatgcacaga gaattcaaac actaccaaat cagaatcagt cgcaaaccca gccattactg 660
aagactcctc ctgctgtact tcagccaatt gcaccacaga caacatttgg tgttcagact 720
cagccccagc cccagtcact gctgcaggca cagatttcag cagcttctat tacaccacta 780
ttgcagactc aaccacagcc cttattacag cagcctcagc aaaaagctgg tttattgcag 840
cctcctgttc gtatagtttc acagccacaa ccggcacgac gattagatcc cccatcccga 900
ttttcaggaa gaaatgacag aggggatcaa gtgcctaaca gaaaagatga tcgaagtcgt 960
gagagagaga gagaaagacg tagatcgaga gaaagatcac ctcagaggaa acgttcccgg 1020
gaaagatctc cacgaagaga gcgagagcga tcacctcgga gagttcgacg tgttgttcca 1080
cgttacacag ttcagttttc aaagttttct ttagattgtc ccagttgtga catgatggaa 1140
ctaaggcgcc gttatcaaaa tttgtatata cctagtgact tttttgatgc tcaatttaca 1200
tgggtggatg ctttcccttt gtcaagacca tttcagctgg gaaattactg caatttttat 1260
gtaatgcaca gagaagtaga gtccttagaa aaaaatatgg ccattcttga tccaccagat 1320
gctgaccact tatacagtgc aaaggtaatg ctgatggcta gccctagtat ggaagattta 1380
tatcataagt catgtgctct tgctgaggac ccacaagaac ttcgagatgg attccaacat 1440
cctgctagac ttgttaagtt tttagtgggc atgaaaggca aggatgaagc tatggccatt 1500
ggaggccact ggtctccttc gttggatgga ccagacccag aaaaagatcc ctctgtgttg 1560
attaagactg ctattcgttg ttgtaaggct ctgacaggca ttgatctaag tgtgtgcaca 1620
caatggtacc gttttgcaga gattcgctac catcgccctg aggagaccca caaggggcgt 1680
acagttccag ctcatgtgga gacagtggtt ttatttttcc cggatgtttg gcattgcctt 1740
cccacccgct cagagtggga aaccctctcc cgaggataca agcagcagct ggtcgagaag 1800
cttcagggtg aacgcaagga ggctgatgga gaacaggatg aagaagagaa ggatgatggt 1860
gaagctaaag aaatttctac acctacccat tggtctaaac ttgatccaaa gacaatgaag 1920
gtaaatgacc tccgaaaaga attagaaagt cgagctctta gttccaaagg attaaaatcc 1980
cagttaatag cccgattgac aaaacagctt aaagtagagg aacaaaaaga agaacagaag 2040
gagttagaga aatctgaaaa agaagaggat gaggatgatg ataggaaatc tgaagacgat 2100
aaagaggaag aagaaaggaa acgtcaagag gaaatagaac gccagcgtcg agaaagaaga 2160
tatattttgc ctgatgaacc ggccatcatt gtacatccaa attgggctgc aaaaagtggc 2220
aagtttgatt gtagcatcat gtctttgagt gtcctattgg actacagatt agaggataat 2280
aaagaacatt catttgaggt ttcattgttt gcggaacttt tcaacgaaat gcttcaaaga 2340
gattttggtg tccgtatata caaatcatta ctgtctcttc ctgagaaaga ggacaaaaaa 2400
gaaaaggata aaaaaagcaa aaaagatgag agaaaagata aaaaagaaga aagagatgat 2460
gaaactgatg aaccaaaacc caaacggaga aaatcaggcg atgataaaga taaaaaagaa 2520
gatagagatg aaaggaagaa agaagataaa agaaaagatg attctaaaga tgatgatgaa 2580
actgaagaag ataacaatca agatgaatat gaccctatgg aagcagaaga agctgaggat 2640
gaagaagatg atagggatga ggaagaaatg accaaacgag atgacaaaag agatatcaac 2700
agatactgca aggagaggcc ctctaaagat aaggaaaaag aaaagactca aatgatcaca 2760
attaacagag atctgttaat ggcttttgtt tattttgatc aaagtcattg tggttacctt 2820
cttgaaaagg atttggaaga aatactttat actcttggac tacatctttc tcgggctcag 2880
gtaaagaagc ttcttaataa agtagtgctc cgtgaatctt gcttttaccg gaaattaaca 2940
gacacctcaa aagatgaaga gaaccatgaa gagtctgagt cattgcagga agatatgcta 3000
ggaaacagat tattacttcc aacaccaaca gtaaagcagg aatcaaagga tgtggaagaa 3060
aatgttggcc tcattgtgta caatggtgca atggtagatg taggaagcct cttgcaaaaa 3120
ttggaaaaga gcgaaaaagt aagagctgag gtagaacaga agctgcagtt actagaagaa 3180
aaaacagatg aagatgaaaa aaccatatta aatttggaga attccaacaa aagcctctct 3240
ggtgaactca gagaagttaa aaaggacctt agtcagttac aagaaaactt aaagatttcg 3300
gaaaacatga atttacaatt tgaaaaccaa atgaataaga caatcagaaa cttatctacg 3360
gtaatggatg aaatccacac tgttctcaag aaggataatg taaagaatga agacaaagat 3420
caaaaatcca aggagaatgg tgccagtgta tga 3453
<210>4
<211>2167bp
<212>DNA
<213>artificial sequence
<220>
<223>
<400>4
atggctcaat ttggaggaca gaagaatccg ccatgggcta ctcagtttac agccactgca 60
gtatcacagc cagctgcact gggtgttcaa cagccatcac tccttggagc atctcctacc 120
atttatacac agcaaactgc attggcagca gcaggcctta ccacacaaac tccagcaaac 180
tatcagttaa cacaaactgc tgcattgcag caacaagccg cagctgcagc agctgcatta 240
caacagcaat attcacaacc tcagcaggcc ctgtatagtg tgcaacaaca gttacagcaa 300
ccccagcaaa ccctcttaac acagccagct gttgcactgc ctacaagcct tagcctgtct 360
actcctcagc caacagcaca aataactgta tcatatccaa caccaaggtc cagtcaacag 420
caaacccagc ctcagaagca gcgtgttttc acaggggtgg ttacaaaact acatgatacg 480
tttggatttg tggatgaaga tgtattcttt cagcttagtg ctgtcaaagg gaaaaccccc 540
caagtaggtg acagagtatt ggttgaagct acttataatc ctaatatgcc ttttaaatgg 600
aatgcacaga gaattcaaac actaccaaat cagaatcagt cgcaaaccca gccattactg 660
aagactcctc ctgctgtact tcagccaatt gcaccacaga caacatttgg tgttcagact 720
cagccccagc cccagtcact gctgcaggca cagatttcag cagcttctat tacaccacta 780
ttgcagactc aaccacagcc cttattacag cagcctcagc aaaaagctgg tttattgcag 840
cctcctgttc gtatagtttc acagccacaa ccggcacgac gattagatcc cccatcccga 900
ttttcaggaa gaaatgacag aggggatcaa gtgcctaaca gaaaagatga tcgaagtcgt 960
gagagagaga gagaaagacg tagatcgaga gaaagatcac ctcagaggaa acgttcccgg 1020
gaaagatctc cacgaagaga gcgagagcga tcacctcgga gagttcgacg tgttgttcca 1080
cgttacacag ttcagttttc aaagttttct ttagattgtc ccagttgtga catgatggaa 1140
ctaaggcgcc gttatcaaaa tttgtatata cctagtgact tttttgatgc tcaatttaca 1200
tgggtggatg ctttcccttt gtcaagacca tttcagctgg gaaattactg caatttttat 1260
gtaatgcaca gagaagtaga gtccttagaa aaaaatatgg ccattcttga tccaccagat 1320
gctgaccact tatacagtgc aaaggtaatg ctgatggcta gccctagtat ggaagattta 1380
tatcataagt catgtgctct tgctgaggac ccacaagaac ttcgagatgg attccaacat 1440
cctgctagac ttgttaagtt tttagtgggc atgaaaggca aggatgaagc tatggccatt 1500
ggaggccact ggtctccttc gttggatgga ccagacccag aaaaagatcc ctctgtgttg 1560
attaagactg ctattcgttg ttgtaaggct ctgacaggca ttgatctaag tgtgtgcaca 1620
caatggtacc gttttgcaga gattcgctac catcgccctg aggagaccca caaggggcgt 1680
acagttccag ctcatgtgga gacagtggtt ttatttttcc cggatgtttg gcattgcctt 1740
cccacccgct cagagtggga aaccctctcc cgaggataca agcagcagct ggtcgagaag 1800
cttcagggtg aacgcaagga ggctgatgga gaacaggatg aagaagagaa ggatgatggt 1860
gaagctaaag aaatttctac acctacccat tggtctaaac ttgatccaaa gacaatgaag 1920
gtaaatgacc tccgaaaaga attagaaagt cgagctctta gttccaaagg attaaaatcc 1980
cagttaatag cccgattgac aaaacagctt aaagtagagg aacaaaaaga agaacagaag 2040
gagttagaga aatctgaaaa agaagaggat gaggatgatg ataggaaatc tgaagacgat 2100
aaagagggat aatgtaaaga atgaagacaa agatcaaaaa tccaaggaga atggtgccag 2160
tgtatga 2167
Claims (4)
1. inhibiting the application of SRSF5 expression and/or active substance in the product that preparation has following (1) or (2) function:
(1) treatment and/or pre- preventing tumor；
(2) inhibit the growth of tumour cell；
The tumour is non-small cell lung cancer；
The inhibition SRSF5 expression and/or active substance are the RNA molecule for interfering SRSF5 expression；
The RNA molecule of the interference SRSF5 expression is to interfere the shRNA of SRSF5 expression；
The shRNA of the interference SRSF5 expression is RNA molecule shown in sequence 1 or sequence 2.
2. application according to claim 1, it is characterised in that: the growth for inhibiting tumour cell is embodied in reduction tumour
The weight of cell and/or the volume of reduction tumour cell and/or the ratio for improving apoptotic tumor cell in situ.
3. application according to claim 1 or 2, it is characterised in that: the inhibition SRSF5 expression and/or active substance
It is that the growth of tumour cell is inhibited by CCAR1 alternative splicing product assay in modulate tumor cell.
4. application according to claim 3, it is characterised in that: CCAR1 alternative splicing product in the modulate tumor cell
Content, which is embodied in, to be improved the content of CCAR1 long form alternative splicing product in tumour cell and/or reduces CCAR1 in tumour cell
CCAR1 long form alternative splicing product and the short shape of CCAR1 in the content and/or raising tumour cell of short-form alternative splicing product
The ratio of formula alternative splicing product.
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| WO2014201413A1 (en) * | 2013-06-14 | 2014-12-18 | Isis Pharmaceuticals, Inc. | Compounds and methods for modulating non-coding rna |
| CN104906575A (en) * | 2014-03-12 | 2015-09-16 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Application of LSECtin as melanoma immunotherapy target |
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| CN101340925A (en) * | 2005-12-22 | 2009-01-07 | 福拉姆斯大学生物技术研究所 | Means and methods for mediating protein interference |
| WO2014201413A1 (en) * | 2013-06-14 | 2014-12-18 | Isis Pharmaceuticals, Inc. | Compounds and methods for modulating non-coding rna |
| CN104906575A (en) * | 2014-03-12 | 2015-09-16 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Application of LSECtin as melanoma immunotherapy target |
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