CN107475299A - The method and its application that research STIM1 genes play a role in non-small cell lung cancer - Google Patents
The method and its application that research STIM1 genes play a role in non-small cell lung cancer Download PDFInfo
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Abstract
The present invention relates to a kind of application of people source STIM1 genes in the cell line proliferations of Lines A549 and SK MES 1 are promoted.The present invention strikes the expression for the endogenous STIM1 for subtracting Lines A549 and SK MES 1 by slow virus, non-small cell lung cancer propagation is promoted to judge STIM1 into the methods of knurl by the detection of MTS propagation, the dyeing of Jim Sa plate clone, flow cytomery cell cycle, nude mice by subcutaneous, as a result show that the propagation and Cell clonality of the cells of Lines A549 and SK MES 1 can be suppressed by striking the expression for subtracting endogenous STIM1, cell-cycle arrest is in the G2/M phases.Experiment of nude mouse shows that growth of the A549 cells in nude mouse can be significantly inhibited by suppressing STIM1 expression, and gross tumor volume is significantly less than control group.
Description
Technical field
The present invention relates to a kind of new method for studying people source STIM1 genes and its application, more particularly to research people source
The method and its application that STIM1 genes play a role in non-small cell lung cancer.
Background technology
Lung cancer is most common lung primary malignant neoplasm, most of to originate from tunica mucosa bronchiorum epithelium.Lung cancer basic pathology
Type is divided into ED-SCLC (SCLC), non-small cell lung cancer (NSCLC), wherein 80% lung cancer belongs to NSCLC.Great mass of data
Show, long-term smoking is an important pathogenic factor of lung cancer, also closely related with the factor such as environment, occupation.Although in recent years
Greater advance is achieved diagnosing and treating, but prognosis is still poor, and its 5 years survival rates are still<15%.Therefore, it is further deep
Enter to study its pathogenesis, find new therapy target, new thinking will be provided for treatment non-small cell lung cancer.
Numerous studies show, in the Carcinogenesis of cell, intracellular message transmission related Ca2+ has abnormal activation
Phenomenon.The calcium ion that the sympathetic albumen 1 of matrix (stromal interaction molecular1, STIM1) manipulates with calcium pond leads to
The activation height correlation in road (Store-Operated Ca2+Channels, SOC), is the focus studied in recent years.
STIM1 genes are located at human chromosomal 11p15.5 regions, and the molecular weight of its encoding proteins is about 77KDa [7].
STIM1 albumen belongs to I type memebrane proteins, has the protein-interacting much predicted and semiotic function area, such as N-terminal signal peptide, EF-hand
Domain, SAM domains, single pass transmembrane (TM) fragment, C-terminal Coiled-coil domains, ERM domains, S/P domains,
Poly-K domain tails etc..
STIM1 is primarily located within endoplasmic reticulum (ER) film.During cell tranquillization, STIM1 is evenly distributed on ER, in ER calcium storehouse
On the basis of exhaustion, the STIM1 being distributed widely in ER is displaced to plasma membrane and ER junctions again, is formed in drain hole, with plasma membrane
Corresponding Orai1 interacts and activates SOC, so as to cause flow of calcium ions.
STIM1 wide expressions in many adults and pediatric tumor, including the nephroblastoma, rhabdomyosarcoma, adrenal gland
Cancer, hepatoblastoma, carcinoma of urinary bladder, lung cancer, oophoroma and carcinoma of testis.However, STIM1 is overexpressed in different cell lines to be caused
Different biological effects:It is capable of inhibiting cell in the bar-shaped tumours of G401 and human rhabdomyosarcoma cells strain to grow and cause cell dead
Die;Cause cell cycle arrest independent of cell differentiation in the muscle fibril cell line of rodent;In breast cancer
Change is then not observed in cell line.It can thus be seen that STIM1 biological effect has its cell type specificity.
In order to study the effect that STIM1 is played in non-small cell lung cancer, the present invention is using the high expression of slow-virus infection
STIM1 Lines A549 and SK-MES-1, with MTS, plate clone dyeing, flow cytometer, nude mice
The subcutaneously influence into detection STIM1 the methods of knurl to lung adenocarcinoma cell propagation and apoptosis.
The content of the invention
An object of the present invention plays a role in a kind of research people source STIM1 genes of offer in non-small cell lung cancer
Method.
The second object of the present invention is in the side to be played a role according to research people source STIM1 genes in non-small cell lung cancer
The conclusion that method is drawn, provide application of the people source STIM1 genes in non-small cell lung cancer propagation.
The third object of the present invention is sequence, slow virus and the host cell for providing research people source STIM1 genes.
The present invention is achieved through the following technical solutions:
A kind of method that research STIM1 genes play a role in non-small cell lung cancer, it comprises the following steps:
1. cultivate Lines A549 and SK-MES-1 cell;
2. use slow virus infected cell, control group is unrelated sequences Scr-shRNA, experimental group STIM1-shRNA-1,
Two sequences of STIM1-shRNA-2, wherein, Scramble sequences are:5 '-TTCTCCGAACGTGTCACGT-3 ', STIM1-
ShRNA-1 sequences are:5 '-CCTGGATGATGTAGATCATAA-3 ', STIM1-shRNA-2 sequence is:5’-
AGAAGGAGCUAGAAUCUCAC-3’.Control group and experimental group carry GFP green fluorescence labels;3. come by the following method
Study effects of the STIM1 in non-small cell lung cancer:
(1) struck with Westren detection slow virus subtract STIM1 strike decreasing effect rate;
(2) struck by MTS detections and subtract ability of cell proliferation after STIM1
(3) struck using plate clone detection and subtract Cell clonality after STIM1;
(4) influence for subtracting cell cycle after STIM1 is struck using flow cytomery.
(5) influence with nude mice by subcutaneous into knurl technology, in vivo layer viewpoint STIM1 gene pairs tumour growth.
As a result show:After striking the STIM1 genes for subtracting Lines A549 and SK-MES-1 cell, cell
Multiplication capacity is remarkably decreased, and cell plates clonality significantly lowers, and cell-cycle arrest is in G2/M phases, nude mouse
Growth of the interior A549 cells in nude mouse is significantly inhibited, and gross tumor volume is significantly less than control group.
According to above experimental result, draw strike subtract people source STIM1 genes suppress Lines A549 and
There is good application in SK-MES-1 cells propagation;Strike and subtract people source STIM1 genes in retardance Lines A549
And have good application in the SK-MES-1 cell cycles;Strike and subtract people source STIM1 genes and promote non-small cell lung cancer in nude mouse
There is good application in tumour growth.
The sequence of research people source STIM1 genes subtracts people source STIMI genes to strike, it is characterised in that:
STIM1-shRNA-1 sequences are:5 '-CCTGGATGATGTAGATCATAA-3 ',
STIM1-shRNA-2 sequences are:5’-AGAAGGAGCUAGAAUCUCAC-3’
Scramble sequences are:5’-TTCTCCGAACGTGTCACGT-3’
The slow virus of research people source STIM1 genes subtracts slow virus used in the STIMI genes of people source to strike, and its feature exists
In:
STIM1-shRNA-1 sequences are:5 '-CCTGGATGATGTAGATCATAA-3 ',
STIM1-shRNA-2 sequences are:5’-AGAAGGAGCUAGAAUCUCAC-3’
Scramble sequences are:5’-TTCTCCGAACGTGTCACGT-3’
The host cell of research people source STIMI genes strikes for use subtracts slow-virus infection used in the STIMI genes of people source
Non-small cell lung cancer A549 and SK-MES-1 cell, it is characterised in that:
STIM1-shRNA-1 sequences are:5 '-CCTGGATGATGTAGATCATAA-3 ',
STIM1-shRNA-2 sequences are:5’-AGAAGGAGCUAGAAUCUCAC-3’
Scramble sequences are:5’-TTCTCCGAACGTGTCACGT-3’
Brief description of the drawings
Fig. 1 is:Slow-virus infection Lines A549 and SK-MES-1 cell, extract albumen, Western
Detect the expression of STIM1 albumen.
Fig. 2 is:MTS detection shows to strike the expression for subtracting STIM1, can significantly inhibit Lines A549 and
The multiplication capacity of SK-MES-1 cells, P<0.05.
Fig. 3 is:Plate clone, which is tested, to be shown to strike the expression for subtracting STIM1, can significantly reduce Lines
The clonality of A549 and SK-MES-1 cells, P<0.01.
Fig. 4 is:Flow cytomery shows to strike the expression for subtracting STIM1, makes Lines A549 and SK-
The cell-cycle arrest of MES-1 cells is in G2/M phases, P<0.05.
Fig. 5 is:Tumor formation in nude mice result shows to strike the expression for subtracting STIM1, can significantly inhibit A549 cells in nude mouse
Interior growth.
Following embodiment will combine above-mentioned accompanying drawing and further illustrate the present invention.
Embodiment
Embodiment 1:STIM1 slow-virus infections
A549 cell lines spread 6 orifice plate after 10 hours, changed the serum-free RPMI-1640 containing 5ug/ulpolybree into
Medium, corresponding viral volume (number of cells * MOI/ virus titers) is added, after cultivating 8 hours, culture medium is changed into and contained
10% hyclone RPMI-1640 Medium are cultivated 72 hours.
Embodiment 2:Western detects protein expression
After infection 72 hours, trypsin digestion cell, complete medium terminates digestion, and the piping and druming of Pasteur's pipe hangs the de- wall of cell
It is floating, cell is collected in 15ml centrifuge tubes after mixing, 1500rpm centrifugation 5min, abandons supernatant;Cell is moved in EP pipes, root
The RIPA (10 μ l/ml protease inhibitors and inhibitors of phosphatases) of suitable volumes is added according to cell concentration, piping and druming mixes, EP pipes
It is put in after sealing membrane closure in 4 DEG C of cell fragmentation Ultrasound Instruments, 500watt, cracks 20min;14000g, 20min are centrifuged, and precipitation is thin
Born of the same parents' fragment, supernatant are cell holoprotein, and BCA methods determine concentration.It is 50ug per PFP applied sample amount.Deposition condition 5% concentrates
Glue 80V, 50 minutes;12% separation gel 120V, 100 minutes.Transferring film condition 100V, 100 minutes.STIM1 primary antibodies dilution ratio 1:
1000, secondary antibody dilution ratio 1:10000.
Shown in testing result (Fig. 1):STIM1 protein expression in experimental group STIM1-shRNA-1, STIM1-shRNA-2
It is substantially lower than untreated fish group and unrelated sequences Scr-shRNA.
Embodiment 3:The expression for subtracting endogenous STIM1 is struck, the increasing of A549 and SK-MES-1 cells can be significantly inhibited
Grow
Struck using MTS experiment detections and subtract endogenous STIM1 expression to Non-small cell lung carcinoma cell line A549 and SK-MES-
The influence of 1 propagation.Take untreated fish group, control group Scr-shRNA and experimental group STIM1-shRNA- in the growth index phase
1st, STIM1-shRNA-2 cells, 96 porocyte culture plates are inoculated in after cell count, per 2000, hole cell, full wavelength scanner
OD values, data analysis are measured at instrument 490nm wavelength.
Experimental result (Fig. 2) is shown:In two plants of cells of A549 and SK-MES-1, with untreated fish group, control group Scr-
ShRNA is compared, and experimental group STIM1-shRNA-1, STIM1-shRNA-2 cell proliferation rate substantially slows down, and difference has system
Meter learns meaning, p<0.05, illustrate that after striking the expression for subtracting endogenous STIM1 Non-small cell lung carcinoma cell A549 can be significantly inhibited
With SK-MES-1 propagation.
Embodiment 4:Strike the expression for subtracting endogenous STIM1, can significantly inhibit non-small cell lung cancer cell A549 and
SK-MES-1 Cell clonality
Experiment is formed using plate clone and subtracts endogenous STIM1 expression to Non-small cell lung carcinoma cell A549 to detect to strike
With the influence of SK-MES-1 clonalities.Take untreated fish group, control group Scr-shRNA and experiment in the growth index phase
Group STIM1-shRAN-1, STIM1-shRAN-2 cell, is inoculated in 6 porocyte culture plates after counting, per 200, hole cell, often
Group sets 3 parallel multiple holes, puts 37 DEG C of 5%CO2Cultivated 14 days or so in constant incubator, cultivate clone's shape visible to naked eyes
Into;After Clone formation, culture being terminated, abandons culture medium, PBS is carefully cleaned 2 times, dried, and adds 1ml methanol to fix 30min,
Abandon after methanol dries and add 1ml Giemsa stains dyeing 30min, wash away dye liquor, dry;Count the clone for being more than 50 cells
Number, does histogram analysis data.
As a result (Fig. 3) is shown:In two plants of cells of A549 and SK-MES-1, experimental group STIM1-shRAN-1, STIM1-
ShRAN-2 cell clonal formations number is substantially fewer than untreated fish group and control group Scr-shRNA, p<0.01.In illustrating to strike and subtracting
After source property STIM1 expression, non-small cell lung cancer cell A549 and SK-MES-1 Cell clonality can be significantly inhibited.
Embodiment 5:The expression for subtracting endogenous STIM1 is struck, non-small cell lung cancer cell A549 and SK-MES- can be made
1 cell-cycle arrest is in the G2/M phases
Endogenous STIM1 expression is subtracted to A549 and SK-MES-1 cell cycles to detect to strike using flow cytometer
Influence.Take the logarithm the cell in growth period, trypsin digestion cell, complete medium terminates, and collects cell in 5ml centrifuge tubes, often
Group sets 3 multiple holes;1200rpm centrifuges 5min, supernatant discarding;PBS (pH7.2-7.4) the washings cell precipitation of 4 DEG C of precoolings 1 time,
1500rpm centrifuges 5min, collects cell;70% ethanol of 4 DEG C of precoolings fixes cell 12h;1500rpm centrifugations 5min goes to fix
Liquid, PBS washings cell precipitation is once;Cell dyeing liquid is prepared:40 × PI mother liquors (2mg/ml):100 × RNase mother liquors (10mg/
ml):1 × PBS=25:10:1000;Cell dyeing:According to cell concentration, 1ml cell dyeing liquids are added, are resuspended slowly and fully thin
Born of the same parents are precipitated, 37 DEG C of lucifuge warm bath 30 minutes.The screen filtration of 300 mesh is in machine pipe in streaming, upper machine testing.Cell leads to during upper machine
It is 200-350cells/s to cross rate;Tested more than in triplicate.
Testing result (Fig. 4) is shown:In two plants of cells of A549 and SK-MES-1, experimental group STIM1-shRAN-1,
STIM1-shRAN-2 cells block in G2/M phases, p compared with untreated fish group and Scr-shRNA groups<0.05, difference has statistics
Meaning.
Case 6 is embodied:The expression for subtracting endogenous STIM1 is struck, life of the A549 cells in nude mouse can be significantly inhibited
Long, gross tumor volume is significantly less than control group
Take in the growth index phase A549 untreated fish groups, control group Scr-shRNA and experimental group STIM1-shRAN-1,
STIM1-shRAN-2 cells, pancreatin are digested to single cell suspension, 2000rpm, centrifuge 5min;PBS liquid is washed twice, abandons supernatant, and
It is resuspended with PBS liquid, cell count, is injected in oxter on the left of 4 weeks BALB/c nude mices of male, every nude mice injects 5*106Individual cell,
Every group of 7 nude mices, nude mice dissected knurl body, arrange data, analysis result into knurl 7 weeks or so.
As a result (Fig. 5) is shown:The tumour growth of experimental group STIM1-shRAN-1, STIM1-shRAN-2, which is considerably slower than, not to be located
Reason group and control group Scr-shRNA, gross tumor volume are significantly less than untreated fish group and control group Scr-shRNA.
Embodiment described above, its description is more specific and detailed, but therefore can not be interpreted as to patent of the present invention
The limitation of scope.It should be pointed out that for the person of ordinary skill of the art, the premise of present inventive concept is not being departed from
Under, various modifications and improvements can be made, these belong to protection scope of the present invention.Therefore, the protection of patent of the present invention
Scope should be determined by the appended claims.
Claims (6)
1. a kind of method that research STIM1 genes play a role in non-small cell lung cancer, it comprises the following steps:
Cultivate Lines A549 cells and SK-MES-1 cells;
Built and packed by slow virus carrier, obtain the virus struck and subtract people source STIMI genes, it is characterised in that:Strike and subtract people source
Lentivirus sequences used in STIMI genes are as follows:STIM1-shRNA-1 sequences (5 '-CCTGGATGATGTAGATCATAA-
3 '), STIM1-shRNA-2 sequences (5 '-AGAAGGAGCUAGAAUCUCAC-3 '), unrelated control Scr-shRNA sequence (5 '-
TTCTCCGAACGTGTCACGT-3’);
Using slow-virus infection non-small cell lung cancer A549 and SK-MES-1 cell, research STIM1 is sent out in non-small cell lung cancer
The effect waved.
2. the method that research STIM1 genes play a role in non-small cell lung cancer as claimed in claim 1,
It is characterized in that:The method for the effect that described research STIM1 is played in non-small cell lung cancer is included in following method
It is at least one:
(1) struck with Westren detection slow virus subtract STIM1 strike decreasing effect rate;
(2) struck by MTS detections and subtract ability of cell proliferation after STIM1
(3) struck using plate clone detection and subtract Cell clonality after STIM1;
(4) influence for subtracting cell cycle after STIM1 is struck using flow cytomery.
(5) influence with nude mice by subcutaneous into knurl technology, in vivo layer viewpoint STIM1 gene pairs tumour growth.
Subtract people source STIMI genes 3. striking, strike and subtract slow virus used in the STIMI genes of people source, and strike and subtract people source STIMI genes
Non-small cell lung cancer A549 and the SK-MES-1 cell of used slow-virus infection.
Subtract people source STIM1 genes answering in Lines A549 and SK-MES-1 cell propagation is suppressed 4. striking
With.
Subtract people source STIM1 genes answering in Lines A549 and the SK-MES-1 cell cycle is blocked 5. striking
With.
Promote application in non-small cell lung cancer tumour growth in nude mouse 6. striking and subtracting people source STIM1 genes.
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Application publication date: 20171215 |