CN109432442A - SLFN5 or SLFN5 promotor is preparing the purposes in lung cancer therapy drug - Google Patents
SLFN5 or SLFN5 promotor is preparing the purposes in lung cancer therapy drug Download PDFInfo
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Abstract
The invention belongs to biological medicines to research and develop field, the purposes in lung cancer therapy drug is being prepared more particularly to SLFN5 or SLFN5 promotor, LFN5 or SLFN5 promotor is one of sole active ingredient or effective component of the lung cancer therapy drug, wherein, lung cancer therapy drug includes SLFN5 the or SLFN5 promotor for imitating dosage；And one of sole active ingredient as lung cancer therapy drug or effective component, SLFN5 refers to using SLFN5 as action target preparing or screen the purposes in lung cancer therapy drug, screens to candidate substances, to find SLFN5 promotor.The present invention has found after extensive and in-depth study, SLFN5 is able to suppress the proliferation of lung carcinoma cell, promote its apoptosis, inhibit its migration, invasion, it is the new target spot and thinking that the treatment of clinically non-small cell lung etc. provides and it was found that SLFN5 is by inhibiting the expression of transcription factor Snail to inhibit the expression of cell membrane anchor type matrix metalloproteinase MT1-MMP to play the effect for inhibiting lung cancer migration and invasion transfer.
Description
Technical field
The invention belongs to biological medicines to research and develop field, and in particular to SLFN5 or SLFN5 promotor is preparing Remedies for lung cancer
Purposes in object.
Background technique
Data show that non-small cell lung morbidity and mortality ranked first in national malignant tumor in 2017
Position, lung cancer seriously threaten human health.It is mainly at present corresponding according to taking for lung cancer by stages for the treatment means of lung cancer
Operation excision, the chemicals such as magnetic target therapy combination cis-platinum of EGFR monoclonal antibody and Chinese medicine are treated.The lethal main original of lung cancer
Because being that cancer cell invasion can not carry out operation excision due to being transferred to other histoorgans.Monoclonal Antibody Against Pulmonary Carcinoma drug acts only at present
Proliferation target spot, therefore find and not only act on proliferation but also while acting on the drug development side that the target drug that invasion are shifted is future
To.SLFN5 gene is the member of Schlafen (SLFN) family, and expression generates protein product.At present about SLFN5 gene
And its function report of albumen is few, is limited primarily to inhibit the activity of immunocyte and inhibits melanoma and kidney hyaline cell
The report of the invasive ability of cancer.The current expression still without SLFN5 in lung cancer and the proliferation for lung cancer, apoptosis,
Invasion, the research of forwarding function report.
Summary of the invention
The technical problem to be solved by the present invention is to promote in view of the deficienciess of the prior art, providing SLFN5 or SLFN5
Agent is preparing the purposes in lung cancer therapy drug, and SLFN5 is used to inhibit the proliferation of lung carcinoma cell, promotes its apoptosis, it is inhibited to move
It moves, invasion, to play the effect for inhibiting lung cancer migration and invasion transfer.To achieve the goals above, the skill that the present invention uses
Art scheme is as follows:
According to an aspect of the present invention, SLFN5 the or SLFN5 promotor provided is preparing the use in lung cancer therapy drug
On the way.
In one embodiment, the lung cancer is primary non-small cell carcinoma, and the lung cancer therapy drug at least has
One of following function:
(1), inhibit the proliferation of lung carcinoma cell；
(2), promote the apoptosis of lung carcinoma cell；
(3), inhibit the migration of lung carcinoma cell；
(4), inhibit the invasion of lung carcinoma cell；
(5), lung carcinoma cell invasion number is reduced；
(6), lung carcinoma cell invasion depth is reduced；
(7), inhibit Snail expression；
(8), inhibit MT1-MMP expression.
Lung cancer therapy drug in above scheme, SLFN5 promotor refer to the substance for improving SLFN5 level.
Specifically, the SLFN5 level that improves can be using the method for various chemistry, physics, biology.
Including but not limited to:
(1a), SLFN5 access is adjusted to improve SLFN5 level；
It is (2a), horizontal in directly increasing SLFN5 in lung carcinoma cell.
SLFN5 or SLFN5 analogies can be delivered in lung carcinoma cell and directly increase SLFN5 level in lung carcinoma cell,
SLFN5 level in lung carcinoma cell can directly be increased by way of being overexpressed SLFN5, adjusting SLFN5 access can be use
SLFN5 agonist is transcribed or is expressed to improve SLFN5 activity or promotion SLFN5, to improve SLFN5 level.Improve SLFN5
Activity is that SLFN5 activity is instigated to improve.Preferably, before compared to improving, SLFN5 activity improves at least 10%, preferably improves extremely
Few 30%, then good raising at least 50%, 70% is more preferably improved, optimal raising at least 90%.
In above scheme, promote SLFN5 transcription or expression to refer to: expressing SLFN5 high, or improves SLFN5 transcription and live
Property, those skilled in the art can be used conventional method and transcribe to SLFN5 or express.
Preferably, SLFN5 transcription or expression at least improve at least 10% to above scheme, preferably raising at least 30%, then
Good raising at least 50% more preferably improves 70%, optimal raising at least 90%.
The embodiment of the present invention is it has proven convenient that by way of being overexpressed SLFN5, and directly SLFN5 is horizontal in increase lung carcinoma cell,
It can inhibit proliferation of lung cancer cells, transfer, invasion, promote Increase Apoptosis of Lung Cancer Cells, treatment lung cancer.And based on the prior art it is found that preceding
SLFN5 level can be raised by stating the method for adjusting SLFN5 metabolic pathway.Thus deduce, the aforementioned side for adjusting SLFN5 metabolic pathway
Method, which also can get, to be inhibited proliferation of lung cancer cells, transfer, invasion, promotes Increase Apoptosis of Lung Cancer Cells, treats the effect of lung cancer, and then is thought
These methods also can inhibit proliferation of lung cancer cells, transfer, invasion, promote Increase Apoptosis of Lung Cancer Cells, treatment lung cancer.
The lung cancer therapy drug necessarily includes SLFN5 or SLFN5 promotor, and with SLFN5 or SLFN5 promotor work
For the effective component of aforementioned function, in the lung cancer therapy drug, the effective component for playing aforementioned function can be only SLFN5
Or SLFN5 promotor, it also may include the molecule that other can play similar function.
Above scheme it is preferred, SLFN5 or SLFN5 promotor be the lung cancer therapy drug sole active ingredient or
One of effective component.
In above scheme, it also can be multi-component compound that the lung cancer therapy drug, which can be single composition substance,.
In above scheme, the form of the lung cancer therapy drug can be solid, liquid, gel, semi-fluid without specifically limited
The various material forms such as matter, aerosol.
In above scheme, the Remedies for lung cancer object mainly for object be mammal, as rodent, spirit are long
Class animal etc..
According to the second aspect of the invention, it to apply SLFN5 or SLFN5 promotor to object, provides in treatment lung
The purposes of cancer, in this embodiment, the lung cancer are primary non-small cell carcinoma；
The object can be mammal.The mammal is preferably rodent, artiodactylous animals, Perissodactyla
Animal, Lagomorph, primate etc., the primate are preferably monkey, ape or people.
The object can be the patient for suffering from lung cancer or expect prevention or alleviate the individual of lung cancer.Or it can be and suffer from
The patient or expectation prevention that suffer from lung cancer or the individual in vitro lung carcinoma cell for alleviating lung cancer.
In this embodiment, SLFN5 or SLFN5 promotor can be applied before, during and after receiving lung cancer therapy to object
With.
According to the third aspect of the invention we, the present invention provides a kind of lung cancer therapy drug, the SLFN5 including effective dose
Or SLFN5 promotor；In this embodiment, the lung cancer is primary non-small cell carcinoma.
In this embodiment, the lung cancer therapy drug include imitate dosage SLFN5 or SLFN5 promotor and
Pharmaceutical carrier.
In this embodiment, the lung cancer therapy drug necessarily includes SLFN5 or SLFN5 promotor, and with SLFN5
Or effective component of the SLFN5 promotor as aforementioned function.
In this embodiment, in the lung cancer therapy drug, the effective component for playing aforementioned function can be only
SLFN5 or SLFN5 promotor also may include the molecule that other can play similar function.
In this embodiment also that is, SLFN5 or SLFN5 promotor be the lung cancer therapy drug it is unique effectively at
Point or one of effective component.
In above scheme, it also can be multi-component compound that the lung cancer therapy drug, which can be single composition substance,.
In above scheme, the form of the lung cancer therapy drug can be solid, liquid, gel, semi-fluid without specifically limited
The various material forms such as matter, aerosol.
In above scheme, the Remedies for lung cancer object mainly for object be mammal, as rodent, spirit are long
Class animal etc..
According to the fourth aspect of the invention, a kind of combination of lung cancer therapy drug is provided, SLFN5 including effective dose or
SLFN5 promotor and other at least one lung cancer therapy drugs, in this embodiment, the lung cancer are that primary is non-small thin
Born of the same parents' cancer, the combination therapy pharmaceutical composition can be any one in following form, and the combination therapy pharmaceutical composition can be with
It is any one in following form:
One) independent preparation, the agent of preparation, is respectively prepared in SLFN5 or SLFN5 promotor and other lung cancer therapy drugs
Type may be the same or different, and administration route also may be the same or different；When other lung cancer therapy drugs are antibody, stomach and intestine are generally used
External administration type.When other lung cancer therapy drugs are chemicals, form of medication can be relatively abundanter, can be gastrointestinal administration
It can also be parenteral administration.It is general that the known administration route for each chemicals is recommended to be administered.
Two) SLFN5 or SLFN5 promotor and other lung cancer therapy drugs, are configured to compound preparation, by SLFN5 or
SLFN5 promotor and other lung cancer therapy drugs can be used using the administration of identical administration route and when applying simultaneously and match the two
It is set to the form of compound preparation.
According to the fifth aspect of the invention, to apply a effective amount of SLFN5 or SLFN5 promotor to object, and to right
As applying other a effective amount of lung cancer therapy drugs and/or implementing the purposes or means of other lung cancer therapies to object, in this kind
In embodiment, the lung cancer is primary non-small cell carcinoma.
In this embodiment, can concurrently or sequentially give a effective amount of SLFN5 or SLFN5 promotor and
Other at least one a effective amount of lung cancer therapy drugs.
In this embodiment, be based on SLFN5 present invention firstly discovers that lung cancer therapy target spot, with SLFN5 or
In other lung cancer therapy combination therapies other than SLFN5 promotor, the effect of curative effect addition can be at least played, further
Enhance the therapeutic effect for lung cancer.
In this embodiment, other lung cancer therapy drugs include but is not limited to: antibody drug, chemicals or target
To type drug etc..
In this embodiment, SLFN5 the or SLFN5 promotor to be to be gastrointestinal administration or parenteral,
Other lung cancer therapy drugs can be gastrointestinal administration or parenteral.
According to the sixth aspect of the invention, provide a kind of SLFN5 or SLFN5 promotor preparation have it is any one of following or
Purposes in the substance of multinomial effect:
(71), SLFN5 or SLFN5 promotor is used to prepare the purposes in the substance for inhibiting proliferation of lung cancer cells；
(72), SLFN5 or SLFN5 promotor is used to prepare the purposes in the substance for promoting Increase Apoptosis of Lung Cancer Cells；
(73), SLFN5 or SLFN5 promotor is used to prepare the purposes in the substance for inhibiting lung carcinoma cell migration；
(74), SLFN5 or SLFN5 promotor is used to prepare the purposes in the substance for inhibiting lung carcinoma cell invasion；
(75), SLFN5 or SLFN5 promotor is used to prepare the purposes in the substance for reducing lung carcinoma cell invasion number；
(76), SLFN5 or SLFN5 promotor is used to prepare the purposes in the substance for reducing lung carcinoma cell invasion depth；
(77), SLFN5 or SLFN5 promotor is used to prepare the purposes in the substance for inhibiting Snail expression；
(78), SLFN5 or SLFN5 promotor is used to prepare the purposes in the substance for inhibiting MT1-MMP expression.
Above-mentioned substance is not necessarily necessary for drug, and in the above-described embodiment, the lung cancer is primary non-small cell
Lung cancer.
According to the seventh aspect of the invention, it provides SLFN5 preparing or screening the purposes in lung cancer therapy drug, in this kind
In embodiment, SLFN5 is as action target, and in this in embodiment, the purposes is specifically referred to: using SLFN5 as work
With target, candidate substances are screened, to find SLFN5 promotor, lung cancer therapy drug alternately.
In conclusion the present invention is due to using above technical scheme, beneficial effect:
The present invention has found that SLFN5 is able to suppress the proliferation of lung carcinoma cell after extensive and in-depth study, it is promoted to wither
It dies, inhibits its migration, invasion, and it was found that SLFN5 is by inhibiting the expression of transcription factor Snail to inhibit cell membrane
The expression of anchor type matrix metalloproteinase MT1-MMP inhibits the effect of lung cancer migration and invasion transfer to play, for clinic
The new target spot and thinking that the treatment of upper lung cancer provides.
Detailed description of the invention
In order to illustrate more clearly of present example or technical solution in the prior art, to embodiment or will show below
There is in technical description required attached drawing do simply to introduce, it is clear that the accompanying drawings in the following description is only of the invention some
Example under the premise of not paying creativeness, can also be obtained according to these attached drawings to those skilled in the art
Other attached drawings.
Figure 1A: Real-time PCR method detection mRNA level in-site ShSLFN5 strikes reduction fruit；
Figure 1B: Western blotting method detection protein level ShSLFN5 strikes reduction fruit；
Fig. 2A: striking with the specific ShRNA (ShSLFN5) of SLFN5 and subtract SLFN5, compared with compareing ShRNA,
ShSLFN5 group cell count all obviously increases on the the 5th, 6,7 day, and there were significant differences for statistical analysis (P < 0.05)；
Fig. 2 B: two groups of proliferation are detected with CCK8 kit, ShCtrl group cell Proliferation is significantly lower than as the result is shown
ShSLFN5 group, there were significant differences for statistical analysis (P < 0.05)；
Fig. 2 C: fluorescence secondary antibody staining analysis, ShCtrl group cell Ki67 is significantly lower than ShSLFN5 group as the result is shown；
Fig. 2 D:Ki-67 immunocytochemical stain: fluorescence secondary antibody staining analysis, as the result is shown ShCtrl group cell Ki67
Significantly lower than ShSLFN5 group, there were significant differences for statistical analysis (P < 0.001)；
Fig. 2 E: cloning experiment detection, ShCtrl group cell Proliferation is significantly lower than ShSLFN5 group as the result is shown；
Fig. 2 F: cloning experiment detection, ShCtrl group cell Proliferation is significantly lower than ShSLFN5 group, statistics as the result is shown
There were significant differences (P < 0.05) for analysis；
Fig. 3 A: building SLFN5 expression vector transfects A549 cell, and screening SLFN5 surely turns cell SLFN5 system (SLFN5),
Compared with transfection control empty carrier (Vector), SLFN5 group cell count is all significantly lower than Vector group, system on the the 5th, 6,7 day
Counting credit analysis, there were significant differences (P < 0.05)；
Fig. 3 B: two groups of proliferation are detected with CCK8 kit, SLFN5 group cell Proliferation is significantly lower than Vector as the result is shown
Group, there were significant differences for statistical analysis (P < 0.05)；
Fig. 3 C: fluorescence secondary antibody staining analysis, SLFN5 group cell Ki67 is significantly lower than Vector group as the result is shown；
Fig. 3 D: fluorescence secondary antibody staining analysis, SLFN5 group cell Ki67 is significantly lower than Vector group, statistics as the result is shown
There were significant differences (P < 0.001) for analysis；
Fig. 3 E: cloning experiment detection, SLFN5 group cell Proliferation is significantly lower than Vector group as the result is shown；
Fig. 3 F: cloning experiment detection, SLFN5 group cell Proliferation is significantly lower than Vector group as the result is shown, counts credit
There were significant differences (P < 0.01) for analysis；
The influence for subtracting SLFN5 to A549 apoptosis is struck in Fig. 4 A:TUNEL detection；
Fig. 4 B:TUNEL detection is overexpressed influence of the SLFN5 to A549 apoptosis；
Fig. 4 C: Flow cytometry strikes the influence for subtracting SLFN5 to A549 apoptosis；
Fig. 4 D: Flow cytometry is overexpressed influence of the SLFN5 to A549 apoptosis；
Fig. 5 A: it strikes and subtracts the groups of cells of SLFN5 and migrate across the cell number of cell film bottom and be apparently higher than control group, statistics
Comparing the two has significant difference (P < 0.05)
Fig. 5 B: Matrigel is it has been observed that strike the cell for subtracting the groups of cells invasion of SLFN5 across Matrigel arrival bottom
Number is apparently higher than control group, and statistics, which compares the two, significant difference (P < 0.01)；
Fig. 5 C: striking the cell for subtracting SLFN5 and be placed on chick chorioallantoic membrane after the preparatory fluorescent staining of cellular control unit,
Frozen section observes that cellular control unit does not invade chorioallantoic membrane, and strike subtract SLFN5 cell (ShSLFN5) invasion across
Chorioallantoic membrane is deep within chicken embryo；
Fig. 5 D: striking the cell for subtracting SLFN5 and be placed on chick chorioallantoic membrane after the preparatory fluorescent staining of cellular control unit,
Frozen section observes that cellular control unit does not invade chorioallantoic membrane, and strike subtract SLFN5 cell (ShSLFN5) invasion across
Chorioallantoic membrane is deep within chicken embryo, and invasion cell number and invasion depth all obviously increase compared with the control group；
Fig. 5 E: the cell for being overexpressed SLFN5 is placed on chick chorioallantoic membrane, and control is observed in frozen section dyeing
Group (Vector) is invaded across chorioallantoic membrane, and the cell for being overexpressed SLFN5 is not invaded across chorioallantoic membrane, with control group
It compares, is overexpressed SLFN5 group cell invasion cell number and invasion depth is all significantly lower than cellular control unit；
Fig. 5 F: the cell for being overexpressed SLFN5 is placed on chick chorioallantoic membrane, observes that control group (Vector) is invaded
The cell attacked across chorioallantoic membrane, and be overexpressed SLFN5 is not invaded across chorioallantoic membrane, and compared with the control group, invasion are thin
Born of the same parents' number and invasion depth are all significantly lower than cellular control unit, and there were significant differences for statistical analysis (P < 0.001)；
Fig. 6 A: discovery SLFN5 has inhibiting effect to Snail expression；
Fig. 6 B: discovery SLFN5 has inhibiting effect to Snail expression；
Fig. 6 C: discovery SLFN5 has inhibiting effect to Snail expression；
Fig. 6 D:SLFN5, which strikes, subtracts the mRNA expression for promoting MT1-MMP；
Fig. 6 E:SLFN5 strikes the expression for subtracting and promoting MT1-MMP albumen；
Fig. 6 F:SLFN5 strikes the expression for subtracting and promoting MT1-MMP albumen；
Fig. 6 G: verifying MT1-MMP siRNA's strikes reduction fruit；
Fig. 6 H: verifying MT1-MMP siRNA's strikes reduction fruit；
Fig. 6 I: verifying MT1-MMP siRNA's strikes reduction fruit；
Fig. 6 J: it is struck in shRNA and subtracts surely turning further to strike in cell line (SLFN5-Sh) and subtract MT1-MMP for SLFN5, found
It strikes and subtracts MT1-MMP and can significantly reduce the transfer ability of A549 cell；
Fig. 6 K: it is struck in shRNA and subtracts surely turning further to strike in cell line (SLFN5-Sh) and subtract MT1-MMP for SLFN5, found
It strikes and subtracts MT1-MMP and can significantly reduce the transfer ability of A549 cell；
Fig. 6 L: it is struck in shRNA and subtracts surely turning further to strike in cell line (SLFN5-Sh) and subtract MT1-MMP for SLFN5, found
It strikes and subtracts MT1-MMP and can significantly reduce the invasive ability of A549 cell；
Fig. 6 M: it is struck in shRNA and subtracts surely turning further to strike in cell line (SLFN5-Sh) and subtract MT1-MMP for SLFN5, found
It strikes and subtracts MT1-MMP and can significantly reduce the invasive ability of A549 cell.
Specific embodiment
Below in conjunction with the attached drawing in present example, technical solution in the embodiment of the present invention carries out clear, complete
Ground description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Based on this
Embodiment in invention, every other reality obtained by those of ordinary skill in the art without making creative efforts
Example is applied, shall fall within the protection scope of the present invention.
SLFN5 or SLFN5 promotor according to the present invention is preparing the purposes in lung cancer therapy drug, present invention research hair
Existing people SLFN5 is able to suppress the proliferation of lung carcinoma cell, promotes its apoptosis, inhibits its migration, invasion, and it was found that SLFN5 is logical
Cross inhibit transcription factor Snail expression so that inhibit cell membrane anchor type matrix metalloproteinase MT1-MMP expression to
Play the effect for inhibiting lung cancer migration and invasion transfer.
Before further describing the specific embodiments of the present invention, it should be appreciated that protection scope of the present invention is not limited to down
State specific specific embodiment；It is also understood that term used in the embodiment of the present invention is specific specific in order to describe
Embodiment, rather than limiting the scope of protection of the present invention.The test method of actual conditions is not specified in the following example,
Usually according to normal condition, or according to condition proposed by each manufacturer.
When embodiment provides numberical range, it should be appreciated that except non-present invention is otherwise noted, two ends of each numberical range
Any one numerical value can be selected between point and two endpoints.Unless otherwise defined, the present invention used in all technologies and
Scientific term is identical as the normally understood meaning of those skilled in the art of the present technique.Except specific method, equipment used in embodiment,
Outside material, grasp and record of the invention according to those skilled in the art to the prior art can also be used and this
Any method, equipment and the material of the similar or equivalent prior art of method described in inventive embodiments, equipment, material come real
The existing present invention.
Unless otherwise stated, disclosed in this invention experimental method, detection method, preparation method be all made of this technology neck
Molecular biology, biochemistry, chromatin Structure and the analysis of domain routine, analytical chemistry, cell culture, recombinant DNA technology and
The routine techniques of related fields.These technologies have perfect explanation in the prior art, and for details, reference can be made to Sambrook etc.
MOLECULAR CLONING " molecular cloning ": A LABORATORY MANUAL, Second edition, Cold Spring
Harbor Laboratory Press, 1989and Third edition, 2001；Ausubel etc., CURRENT
PROTOCOLS IN MOLECULAR BIOLOGY " modern molecular biology common method ", John Wiley&Sons, New
York, 1987and periodic updates；" the enzymology side the series METHODS IN ENZYMOLOGY
Method ", Academic Press, San Diego；Wolffe, CHROMATIN STRUCTURE AND FUNCTION " chromatin knot
Structure and function ", Third edition, Academic Press, San Diego, 1998；METHODS IN ENZYMOLOGY
" enzymology method ", Vol.304, Chromatin " chromatin " (P.M.Wassarman and A.P.Wolffe, eds.),
Academic Press, San Diego, 1999；With " the molecular biology research side METHODS IN MOLECULAR BIOLOGY
Method ", Vol.119, Chromatin Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc..
1 method of embodiment
1, cell culture
Lung cancer cell line A549 is purchased from U.S. ATCC.DMEM high glucose medium, 10%FBS (are purchased from Chinese holly company),
100U ampicillin, 100 μ g/ml streptomysins, 37 DEG C, 5%CO2 incubator culture.
2, RT-PCR and realtime-PCR method
Cell total rna is extracted with Trizol, two-step method reverse transcription synthesizes cDNA.Special primer is designed, using SYBR
Green PCR Master Mix (Applied Biosystems) carries out Real-time PCR and measures each gene expression dose,
RT-PCR and 2% agarose gel electrophoresis carry out product length and single band specificity verification, as shown in table 1.
Table 1
3、Western Blot
Total protein of cell is extracted, conventional SDS-PAGE protein isolate band turns nitrocellulose filter, 5% skimmed milk power envelope
It closes, 4 DEG C of primary antibody are incubated overnight, act within secondary antibody room temperature 1 hour, ECL reagent chemiluminescence, exposure, qualitative point of protein band
Analysis, semi-quantitative analysis.Primary antibody: anti-SLFN5 (sigma) 1:2,000, anti-snail (Santacluz), anti-MT1-
MMP(abcam).Secondary antibody couples anti-mouse or anti-rabbit antibody (cellsignalling with corresponding HRP
technology)。
4, shRNA is transfected
The special shRNA sequence of design synthesis people SLFN5 (NM144975.3):
5`-AUUCGUUGGAGUCUGAGUC-3` (SEQ ID NO.7), it is complementary with the region 535-537 in sequence.GC contains
Measuring identical shRNA is negative control, and X-tremeGENE HP (Roche) transfects cell.It is full culture medium that cell, which changes liquid, after 4h,
Real-time PCR detection SLFN5knockdown strikes reduction fruit, carries out subsequent molecular biology and function assessment experiment.
5, slow virus carrier building and A549 surely turn cell line building
Cell culture, extracts total serum IgE, and reverse transcription obtains cDNA.Referring to NM144975.3 primers, PCR method
Amplification obtains the open reading frame of SLFN5, upstream primer sequence are as follows: 5 '-GAGGATCCCCGGGTACCGGTCGCCACCAT
GAGTCTTAGGATTGATGT GGATAC-3'(SEQ ID NO.8)；
Downstream primer sequence are as follows:
5 '-TCCTTGTAGTCCATACCCACAGAAGCCTTCAGAATATACAGATG -3 ' (SEQ ID NO.9), PCR
Primer size 2717bp.
PCR product be connected in Lenti-virus carrier GV358 carrier through AgeI/AgeI digestion (Vector map:
Http:// www.genechem.com.cn/Zaiti.aspx? zt=GV358).
Carrier element sequence Ubi-MCS-3FLAG-SV40-EGFP-IRES-puromycin, eGFP original part band independent startup
Son.293T cell is transfected, with the expression of fluorescence microscope verifying recombinant vector, cell protein is extracted and carries out Western
Blot verifies the specific expressed of SLFN5.4th day collection culture medium supernatant after transfection carries out virus titer mirror after ultrafiltration concentration
Fixed, titre reaches 108It can be used for testing.
Human lung cancer cell line A549 cell line is cultivated, transfects Lenti-SLFN5 slow virus and viral blank control respectively,
The screening of EGFP streaming, constructs the cell line for stablizing expression SLFN5 and empty carrier.Anti-SLFN5 (Sigma) primary antibody, fluorescence two
Anti-dye, fluorescence microscope express the cell percentages of SLFN5.Real-time PCR and Western blot is carried out, point
Do not further determine that stably transfected cell line constructs successfully from mRNA level in-site and protein level.
6, cellular immunofluorescence
4% paraformaldehyde is fixed, 0.1%Triton X-100 permeable membrane, and 5% lowlenthal serum and 3%BSA closing are used respectively
Primary antibody anti-SLFN5, anti-Ki67, anti-MT1-MMP are incubated at room temperature 2 hours, and Alexa 488 or Alexa 594 are marked
Fluorescence secondary antibody (Invitrogen) be incubated at room temperature 1 hour, DAPI redyes nucleus, fluorescence microscope or laser confocal scanning
Microscopy results.
7, CCk-8 proliferation detection
100 μ l2000 cells are added in the every hole of 96 orifice plates, and 10 μ lCCK-8 solution (the green skies) are added in every hole.It is not added
The hole of cell is as blank control.Continue to be incubated for 2 hours in cell incubator, detects 450nm absorbance with microplate reader.
8, TUNEL is detected
Cells rinsed with PBS, 4% paraformaldehyde fixed cell 30 minutes, the PBS room temperature of 0.3%Triton X-100 was incubated
It educates 5 minutes.On sample plus 50 μ lTUNEL detect liquid (the green skies), and 37 DEG C are protected from light incubation 60 minutes.PBS or HBSS washing 3
It is secondary.Under the microscope with fluorescence microscopy after anti-fluorescent quenching mounting fluid-tight piece.The excitation wavelength of Cy3 is 550nm, and launch wavelength is
570nm (red fluorescence).
9, the migration of the cell Transwell and Matrigel
Matrigel: preparation matrigel matrigel and DMEM mixed system, ratio 1:3 are operated on ice, each cell
Be added in upper chamber 100 μ l premix system, cell is put into 24 orifice plates and is placed in 37 DEG C of constant temperature incubators, after 4h to its solidify into
Row is tested in next step, is tested in next step consistent with migration experimental procedure.
Migration experiment: digesting A549 cell with pancreatin, after rinsing cell three times with the DMEM of no FBS, utilizes cell count
Plate carries out cell count.Cell is rinsed with the DMEM of no FBS (to buy from corning) 3 times, small interior addition cell 5x105,
The 100 μ l of DMEM culture medium of 15%FBS is added into lower chambers.24 orifice plates for being loaded with the cell transwell are put into 37 degree to incubate
In case, place for 24 hours.It takes out transwell and cleans cell 3 times with PBS, with fixer (the green skies) fixed 10min.It absorbs solid
Determine liquid, clean cell 3 times with PBS, dyes 5min with crystal violet (the green skies).Crystal violet is sucked, PBS cleans cell 3 times, uses
Cotton ball soaked in alcohol wipes the cell on surface.Observation shooting is carried out under microscope.
10, Transfer Experiment is invaded in chicken embryo body
The cell seeding of fluorescent marker is placed 3 days in chicken embryo chorioallantoic membrane upper surface, incubator.It terminates real
It tests, 4% paraformaldehyde fixed sample.The dyeing of IV Collagen Type VI antibody, DAPI redye nucleus, and fluorescence microscopy under the microscope, is taken a picture
Record is as a result, statistical analysis invasion cell quantity and invasion depth.
2 result of embodiment
1, ShRNA, which strikes, subtracts SLFN5 and surely turns cell line and strike to subtract compliance test result, transfects A549 with SLFN5 special ShRNA
Cell and screening surely turns cell line (ShSLFN5), while the cell construction of transfection control ShRNA surely turns cell line (ShCtrl):
Real-time PCR detection mRNA level in-site strikes reduction fruit (such as Figure 1A)；Western blotting detection protein level strikes reduction
It is all more satisfactory that fruit (such as Figure 1B), mRNA level in-site and protein level strike reduction fruit, can be used for subsequent experimental research.
2, SLFN5 ShRNA strikes the proliferation for subtracting and testing and finding that SLFN5 can inhibit lung cancer cell line A549:
(1) method for cell count: striking with the specific ShRNA (ShSLFN5) of SLFN5 and subtract SLFN5, and compares ShRNA
It compares, ShSLFN5 group cell count, all obviously increases within the 5th, 6,7 day, there were significant differences for statistical analysis (P < 0.05), such as
Shown in Fig. 2A.
(2) CCK8 method is tested: detecting two groups of proliferation with CCK8 kit, as the result is shown ShCtrl group cell Proliferation
Significantly lower than ShSLFN5 group, there were significant differences for statistical analysis (P < 0.05), as shown in Figure 2 B.
(3) Ki-67 immunocytochemical stain: fluorescence secondary antibody staining analysis, ShCtrl group cell Ki67 is bright as the result is shown
Aobvious to be lower than ShSLFN5 group, there were significant differences for statistical analysis (P < 0.001), as shown in Fig. 2 C and Fig. 2 D；ShCtrl group Ki-67
Dyeing is very weak, indicates that ShCtrl group Ki67 expression quantity is significantly lower than ShSLFN5 group, ShCtrl group ability of cell proliferation is weak, explanation
SLFN5 is able to suppress proliferation of lung cancer cells.It is very weak to scheme ShCtrl group Ki-67 dyeing, indicates that ShCtrl group Ki67 expression quantity is obvious
Lower than ShSLFN5 group, ShCtrl group ability of cell proliferation is weak, illustrates that SLFN5 is able to suppress proliferation of lung cancer cells.
(4) cell clone is tested: cloning experiment detection, ShCtrl group cell Proliferation is significantly lower than as the result is shown
ShSLFN5 group, there were significant differences for statistical analysis (P < 0.05), as shown in Fig. 2 E and Fig. 2 F.
3, the proliferation of lung cancer cell line A549 can be inhibited by being overexpressed SLFN5 discovery SLFN5:
(1) method for cell count: building SLFN5 expression vector transfects A549 cell, and screening SLFN5 surely turns cell
SLFN5 system (SLFN5), compared with transfection control empty carrier (Vector), SLFN5 group cell count, the 5th, 6,7 day all obvious low
In Vector group, there were significant differences for statistical analysis (P < 0.05), as shown in Figure 3A.
(2) CCK8 method is tested: detecting two groups of proliferation with CCK8 kit, SLFN5 group cell Proliferation is bright as the result is shown
Aobvious to be lower than Vector group, there were significant differences for statistical analysis (P < 0.05), such as Fig. 3 B.
(3) Ki-67 immunocytochemical stain: fluorescence secondary antibody staining analysis, SLFN5 group cell Ki67 is obvious as the result is shown
Lower than Vector group, there were significant differences for statistical analysis (P < 0.001), as shown in Fig. 3 C and Fig. 3 D；By expressing SLFN5 group
Ki-67 dyeing is very weak, hence it is evident that is lower than vehicle Control group, illustrates that SLFN5 inhibits the proliferation of cell.
(4) cell clone is tested: cloning experiment detection, SLFN5 group cell Proliferation is significantly lower than Vector as the result is shown
Group, there were significant differences for statistical analysis (P < 0.01), as shown in Fig. 3 E and Fig. 3 F.
4, SLFN5 promotes the apoptosis of lung cancer cell line A549:
(1) TUNEL dyeing: nucleus is dyed with DAPI, and apoptotic cell TUNEL dyeing is red, and SLFN5, which strikes, subtracts group carefully
Born of the same parents TUNEL dyeing be significantly lower than cellular control unit, there were significant differences for statistical analysis (as shown in Figure 4 A, ShCtrl group cell
The white bright spot that TUNEL dyeing is presented is apoptosis positive staining, and ShSLFN5 group cell has no that white bright spot shows apoptosis yin
Property).And the TUNEL dyeing for being overexpressed SLFN5 group cell is above empty vector control group, there were significant differences for statistical analysis (such as
Shown in Fig. 4 B).
(2) flow cytometer showed: SLFN5 strikes the early apoptosis rate for subtracting group cell and late apoptic rate is below cellular control unit,
There were significant differences for statistical analysis (as shown in Figure 4 C).And it is overexpressed the early apoptosis rate and late apoptic rate of SLFN5 group cell
It is above empty vector control group, there were significant differences for statistical analysis (as shown in Figure 4 D).
5, SLFN5 inhibits the migration of lung cancer cell line A549, invasive ability:
(1) it strikes and subtracts the migration experiment of the cell SLFN5: striking and subtract the groups of cells of SLFN5 and migrate across the cell number of cell film bottom
It is apparently higher than control group, statistics, which compares the two, significant difference (P < 0.05), as shown in Figure 5A.
(2) strike and subtract SLFN5 Matrigel: Matrigel it has been observed that strike subtract the groups of cells invasion of SLFN5 across
The cell number that Matrigel reaches bottom is apparently higher than control group, and statistics, which compares the two, to be had significant difference (P < 0.01), such as
Shown in Fig. 5 B.
(3) it strikes and subtracts Matrigel in SLFN5 chicken embryo body: striking and subtract the cell (green fluorescence label) of SLFN5 and be placed on chicken embryo
In chorioallantoic membrane, frozen section, H&E is dyed and DAPI nuclear targeting, observes that control group does not invade chorioallantoic membrane, and
Cell (ShSLFN5) invasion for subtracting SLFN5 are struck across chorioallantoic membrane, are deep within chicken embryo, invasion cell number and invasion are deep
Degree all obviously increases compared with the control group, and there were significant differences for statistical analysis (P≤0.001), as shown in Fig. 5 C and Fig. 5 D.
(4) it is overexpressed SLFN5 and inhibits Matrigel in chicken embryo body, the cell (green fluorescence label) for being overexpressed SLFN5 is put
It sets on chick chorioallantoic membrane, frozen section, H&E dyeing and DAPI nuclear targeting observe that control group (Vector) is invaded
The cell attacked across chorioallantoic membrane, and be overexpressed SLFN5 is not invaded across chorioallantoic membrane, and compared with the control group, invasion are thin
Born of the same parents' number and invasion depth are all significantly lower than cellular control unit, and there were significant differences for statistical analysis (P < 0.001), such as Fig. 5 E and Fig. 5 F
Shown, in the vehicle Control group in Fig. 5 E, white arrow instruction invasion enter the lung carcinoma cell inside chicken embryo, and SLFN5 group
Lung carcinoma cell has no that invasion enter inside chicken embryo, shows that SLFN5 can inhibit to invade.
6, have found that SLFN5 inhibits the mechanism of invasion:
(1) Snail is the transcription factor that regulating and controlling effect is played in lung cancer A549 cell invasion transferring path, it has been found that
SLFN5 has inhibiting effect to Snail expression (as shown in Fig. 6 A, Fig. 6 B and Fig. 6 C).
(2) Snail inhibits the expression of cell anchor type matrix metalloproteinase MT1-MMP as transcription factor.MT1-MMP
It is distributed on cell membrane, the matrix components for cell peripheral environment of degrading enable tumour cell to migrate and invade transfer.It is high
Expression be tumor cell invasion transfer directly facilitate factor.We have discovered that SLFN5 inhibits MT1-MMP expression (experiment number
According to as shown in Fig. 6 J, Fig. 6 K, Fig. 6 L and Fig. 6 M), the mechanism for showing that SLFN5 inhibits lung cell A549 invasion transfer is to pass through suppression
The expression of Snail processed, and then the expression for the downstream albumen MT1-MMP for inhibiting it to regulate and control, to reach inhibition tumor cell invasion
The effect of transfer.
(3) experimental data is as follows: SLFN5, which strikes, subtracts the mRNA expression (as shown in Figure 6 D) for promoting MT1-MMP and protein table
Up to (as shown in Fig. 6 E and Fig. 6 F)；Verifying MT1-MMP siRNA's strikes reduction fruit (Fig. 6 G, Fig. 6 H, Fig. 6 I)；It strikes and subtracts in shRNA
SLFN5's surely turns further to strike in cell line (SLFN5-Sh) and subtracts MT1-MMP, and discovery, which is struck, to be subtracted MT1-MMP and can significantly reduce A549
The migration (as shown in Fig. 6 J and Fig. 6 K) of cell and invasive ability (as shown in Fig. 6 L and Fig. 6 M).
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in the use of the new type
Spirit and principle within, any modification, equivalent replacement, improvement and so on should be included within the protection scope of invention.
Claims (10)
1.SLFN5 or SLFN5 promotor is preparing the purposes in lung cancer therapy drug.
2. purposes according to claim 1, which is characterized in that the lung cancer therapy drug at least have following function it
One:
(1), inhibit the proliferation of lung carcinoma cell；
(2), promote the apoptosis of lung carcinoma cell；
(3), inhibit the migration of lung carcinoma cell；
(4), inhibit the invasion of lung carcinoma cell；
(5), lung carcinoma cell invasion number is reduced；
(6), lung carcinoma cell invasion depth is reduced；
(7), inhibit Snail expression；
(8), inhibit MT1-MMP expression.
3. purposes according to claim 1, which is characterized in that SLFN5 or SLFN5 promotor is the lung cancer therapy drug
Sole active ingredient or one of effective component.
4. a kind of lung cancer therapy drug, SLFN5 the or SLFN5 promotor including effective dose.
5. lung cancer therapy drug according to claim 4, which is characterized in that SLFN5 or SLFN5 promotor is the lung cancer
One of the sole active ingredient of therapeutic agent or effective component.
6. a kind of lung cancer therapy drug combination, SLFN5 or SLFN5 promotor and other at least one lung cancer including effective dose
Therapeutic agent.
7.SLFN5 or SLFN5 promotor has the purposes in any one of following or multinomial effect substance in preparation:
(71), SLFN5 or SLFN5 promotor is used to prepare the purposes in the substance for inhibiting proliferation of lung cancer cells；
(72), SLFN5 or SLFN5 promotor is used to prepare the purposes in the substance for promoting Increase Apoptosis of Lung Cancer Cells；
(73), SLFN5 or SLFN5 promotor is used to prepare the purposes in the substance for inhibiting lung carcinoma cell migration；
(74), SLFN5 or SLFN5 promotor is used to prepare the purposes in the substance for inhibiting lung carcinoma cell invasion；
(75), SLFN5 or SLFN5 promotor is used to prepare the purposes in the substance for reducing lung carcinoma cell invasion number；
(76), SLFN5 or SLFN5 promotor is used to prepare the purposes in the substance for reducing lung carcinoma cell invasion depth；
(77), SLFN5 or SLFN5 promotor is used to prepare the purposes in the substance for inhibiting Snail expression；
(78), SLFN5 or SLFN5 promotor is used to prepare the purposes in the substance for inhibiting MT1-MMP expression.
8.SLFN5 is preparing or is screening the purposes in lung cancer therapy drug.
9. purposes according to claim 8, which is characterized in that SLFN5 is as action target.
10. purposes according to claim 8, which is characterized in that the purposes specifically refers to: using SLFN5 as effect target
Mark, screens candidate substances, to find SLFN5 promotor, lung cancer therapy drug alternately.
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