CN103773802B - HIP-55 albumen suppresses the application in tumour medicine in exploitation - Google Patents
HIP-55 albumen suppresses the application in tumour medicine in exploitation Download PDFInfo
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Abstract
The invention discloses the new application of a kind of HIP-55 albumen. Recombinant vector provided by the invention, for the DNA molecular of coded interference HIP-55 protein expression siRNA is inserted expression vector, obtains recombinant vector; The aminoacid sequence of described HIP-55 albumen is the sequence 2 in sequence table. Additionally provide HIP-55 albumen or have following 1 containing the recombinant vector of HIP-55 protein coding gene in preparation) and/or 2) application in the product of function: 1) promote tumor cell migration; 2) tumor cell invasion is promoted; The aminoacid sequence of described HIP-55 albumen is the sequence 2 in sequence table. The experiment proves that, by the gene of this albumen of process LAN in lung carcinoma cell, then promote tumor to be formed, promote that lung carcinoma cell migrates and lung carcinoma cell invasion; Therefore, HIP-55 is that clinical diagnosis, treatment and new drug development have carried for new target spot, it is possible to at screening, exploitation and/or design prevention and/or treatment tumor product.
Description
Technical field
The present invention relates to biological technical field, particularly relate to a kind of HIP-55 albumen and suppress the application in tumour medicine in exploitation.
Background technology
HIP-55 (hematopoieticprogenitorkinase1 [HPK1]-interactingproteinof55kDa; Also referred to as drebrin-likeproteinDBNL, mAbp1 or SH3P7) it is the protein comprising multi-functional territory. Its structure mainly comprises F-actin protein binding domain and the C end SH3 domain of N end. HIP-55 participates in synapse generation, receptor endocytosis, cytoskeleton rearrangement and signal transduction. At present HIP-55 function is understood less, it is known that HIP-55 is presented etc. by regulatory T-cell receptor endocytosis, release of cytokines and B-cell receptor and plays an important role in immune system. Recent research indicate that HIP-55 regulates cytoskeleton rearrangement by interacting with N-WASP and Arp2/3 in nervous system. But its effect in tumor all there is no research.
Summary of the invention
It is an object of the present invention to provide a kind of recombinant vector.
Recombinant vector provided by the invention, for the DNA molecular of coded interference HIP-55 protein expression siRNA is inserted expression vector, obtains recombinant vector; The aminoacid sequence of described HIP-55 albumen is the sequence 2 in sequence table.
In above-mentioned recombinant vector, the nucleotides sequence of the DNA molecular of described coded interference HIP-55 protein expression siRNA is classified as in sequence table sequence 5. In an embodiment of the present invention, described expression vector is pSilencer5.1; Recombinant vector is pSilencer5.1-siRNA, between BamHI and the HindIII restriction enzyme site by DNA molecular (sequence 5) the insertion pSilencer5.1 carrier of coding siRNA, obtains the carrier of the HIP-55siRNA shown in expressed sequence 3.
Above-mentioned recombinant vector has following 1 in preparation)-7) at least one function product in application be also the scope of protection of the invention:
1) cell proliferation is suppressed;
2) apoptosis is promoted;
3) tumor cell migration is suppressed;
4) tumor cell invasion is suppressed;
5) tumor cell clone is suppressed to be formed;
6) tumor is suppressed to be formed;
7) prevention and/or treatment tumor.
In above-mentioned application, described product is medicine.
In above-mentioned application, described tumor is pulmonary carcinoma; Described tumor cell is tumor cell.
In above-mentioned application, described tumor cell is lung carcinoma cell; Described lung carcinoma cell is specially Non-small cell lung carcinoma cell.
It is a further object to provide HIP-55 albumen or its encoding gene or the application of the recombinant vector containing HIP-55 protein coding gene.
HIP-55 albumen provided by the invention or its encoding gene or the recombinant vector containing HIP-55 protein coding gene have following 1 in preparation) and/or 2) application in the product of function:
1) tumor cell migration is promoted;
2) tumor cell invasion is promoted;
The aminoacid sequence of described HIP-55 albumen is the sequence 2 in sequence table.
In above-mentioned application, the nucleotides sequence of described HIP-55 protein coding gene is classified as the sequence 1 in sequence table.
In above-mentioned application, described product is medicine.
In above-mentioned application, described tumor is pulmonary carcinoma;
Described tumor cell is tumor cell; Described tumor cell is lung carcinoma cell; In an embodiment of the present invention, described lung carcinoma cell behaviour non-small cell lung cancer cell, it is specially Non-small cell lung carcinoma cell A549.
The application in screening, exploitation and/or design prevention and/or treatment tumor product of the HIP-55 albumen is also the scope of protection of the invention; The aminoacid sequence of described HIP-55 albumen is the sequence 2 in sequence table.
The experiment proves that, present invention discover that HIP-55 obvious high expressed in clinical lung cancer patient tissue samples, further study showed that the function of HIP-55: by the gene of this albumen of process LAN in lung carcinoma cell, it is possible to promote tumor to be formed, promote that lung carcinoma cell migrates and lung carcinoma cell invasion; By the reticent HIP-55 albumen of RNA interference, it is possible to substantially suppress tumor to be formed, promote apoptosis, Inhibit proliferaton, cell clonal formation, cell migration and/or cell invasion, can be used to prepare prevention and/or treatment tumor product. Therefore, HIP-55 is that clinical diagnosis, treatment and new drug development have carried for new target spot, it is possible to at screening, exploitation and/or design prevention and/or treatment tumor product.
Accompanying drawing explanation
Fig. 1 be RNA disturb HIP-55 cell in protein expression
Fig. 2 be process LAN HIP-55 cell in protein expression
Fig. 3 is the RNA cell proliferation disturbing HIP-55
Fig. 4 is the cell proliferation of process LAN HIP-55
Fig. 5 is the HIP-55 impact on lung cell A549 apoptosis
Fig. 6 is the HIP-55 impact on apoptosis markers
Fig. 7 is the HIP-55 impact on Cell clonality
Fig. 8 is the impact of scratch test detection HIP-55 on cell migration
Fig. 9 is the Transwell impact detecting HIP-55 on cell migration
Figure 10 is that cell invasion is affected by HIP-55
Figure 11 is the HIP-55 impact on becoming tumor
Figure 12 is HIP-55 high expressed in patients with lung cancer tissue samples
Detailed description of the invention
The experimental technique used in following embodiment if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, all commercially obtain.
In following embodiment, quantitative experiment is all in triplicate, results averaged.
In following embodiment, Anti-HIP-55 antibody is purchased from BDBioscience (SanDiego, CA).
Non-small cell lung carcinoma A549(U.S. ATCC, catalog number CCL-185 in following embodimentTM) containing 10% hyclone, 100U/ml penicillin, 100U/ml streptomycin DMEM in high glucose culture fluid in cultivate, and be placed on 37 DEG C, in the CO2 gas incubator of 5% concentration. A549 cell is through had digestive transfer culture, and trophophase cell of taking the logarithm is stand-by.
The aminoacid sequence of people's HIP-55 albumen is the sequence 2 in sequence table, encodes the sequence 1 that the nucleotides sequence of the gene of this albumen is classified as in sequence table.
Embodiment 1, the A549 cell of process LAN HIP-55 and strike the structure of the A549 cell subtracting HIP-55
One, the acquisition of HIP-55siRNA
Following siRNA sequence is designed and synthesized for people's HIP-55 protein coding gene:
HIP-55siRNA, its nucleotides sequence is classified as 5'-GAUCCGCAGUGAACGUAGAGAAUUGUUCAAGAGACAAUUCUCUACGUUCACUG UUUUUUGGAAA-3'(sequence 3);
Comparison ScramblesiRNA, its nucleotides sequence is classified as 5'-GAUCCGCACUACCGGUUGUAUAGGUGUUCAAGAGACACCUAUACAAAGGUAGU GUUUUGGAAA-3'(sequence 4).
Two, the recombinant vector of process LAN HIP-55 and RNA disturb the structure of the recombinant vector of HIP-55
1, the recombinant vector pLenti6/V5-HIP-55 of process LAN HIP-55 builds
In above-mentioned sequence table, the gene of the HIP-55 shown in sequence 1 is for template, uses
Forward: '-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGCGGCGAACCTGAGCC-3 ' and
Reverse:5 '-GGGGACCACTTTGTACAAGAAAGCTGGGTCCTCAATGAGCTCCACGTAGT-3 ' primer expands, and obtains the PCR primer of 1356bp.
By the PCR primer of 1356bp with pDONR221 carrier (purchased from Invitrogen, catalog number is 12536017) carry out BP reaction, obtain recombinant vector pDONR221-HIP-55, again by pDONR221-HIP-55 and pLenti6/V5-DEST (purchased from Invitrogen, catalog number is V49610X) carry out LR reaction, obtain recombinant vector pLenti6/V5-HIP-55. Through order-checking, the DNA molecular shown in the sequence 1 in sequence table is inserted the carrier obtained between the pLenti6/V5-DEST of expression vector by this recombinant vector pLenti6/V5-HIP-55.
2, RNA disturbs the structure of the recombinant vector pSilencer5.1-siRNA of HIP-55
The DNA molecular of composite coding HIP-55siRNA, its nucleotides sequence is classified as; 5'-GATCCGCAGTGAACGTAGAGAATTGTTCAAGAGACAATTCTCTACGTTCACTG TTTTTTGGAAA-3'(sequence 5); Being formed by the annealing of two single stranded DNAs, wherein the nucleotides sequence of a single stranded DNA is classified as the nucleotides sequence of F:5'-GATCCGCAGTGAACGTAGAGAATTGTTCAAGAGACAATTCTCTACGTTCAC another single stranded DNA of TGTTTTTTGGAAA-3' and is classified as R:5'-AGCTTTTCCAAAAAACAGTGAACGTAGAGAATTGTCTCTTGAACAATTCTC TACGTTCACTGCG-3'.
The DNA molecular (sequence 5) of coding HIP-55siRNA is inserted pSilencer5.1(Invitrogen by rna interference vector pSilencer5.1-siRNA, AM5782), between BamHI and the HindIII restriction enzyme site of carrier, the carrier of the HIP-55siRNA shown in expressed sequence 3 is obtained.
This DNA molecular is made up of forward DNA fragmentation, stuffer fragment and inverted DNA fragment; Wherein forward DNA fragmentation be in sequence table sequence 5 from 5 ' end 7-25 position nucleotide, stuffer fragment be in sequence table sequence 5 from 5 ' end 26-34 position nucleotide and inverted DNA fragment be in sequence table sequence 5 from the DNA molecular shown in 5 ' end 35-53 position nucleotide.
Comparison rna interference vector pSilencer5.1-Scramble: between BamHI and the HindIII site by the DNA molecular insertion pSilencer5.1 carrier of coding ScramblesiRNA, obtain the carrier of the ScramblesiRNA shown in expressed sequence 4.
The DNA molecular of above-mentioned coding ScramblesiRNA, its nucleotides sequence is classified as 5'-GATCCGCACTACCGGTTGTATAGGTGTTCAAGAGACACCTATACAAAGGTAGT GTTTTGGAAA-3'(sequence 6); This DNA molecular is made up of forward DNA fragmentation, stuffer fragment and inverted DNA fragment; Wherein forward DNA fragmentation be in sequence table sequence 6 from 5 ' end 7-25 position nucleotide, stuffer fragment be in sequence table sequence 6 from 5 ' end 26-34 position nucleotide and inverted DNA fragment be in sequence table sequence 6 from 5 ' end 35-53 position nucleotide.
Three, the A549 cell of process LAN HIP-55 and RNA disturb the structure of the A549 cell of HIP-55
1, the structure of the A549 cell of process LAN HIP-55
The recombinant vector pLenti6/V5-HIP-55 obtained above-mentioned two infects in A549 cell, obtains A549/pLenti6/V5-HIP-55.
2, the structure of the A549 cell of process LAN HIP-55 is compareed
Expression vector pLenti6/V5-DEST is infected in A549 cell, obtains A549/pLenti6/V5.
3, RNA disturbs the structure of the A549 cell of HIP-55
In the rna interference vector pSilencer5.1-siRNA liposome transfection A549 cell obtained above-mentioned two, puromycin (puromycin, 1.25 �� g/ml) screens viral infection positive cell clone, obtains A549/pSilencer5.1-siRNA.
4, comparison RNA disturbs the structure of A549 cell
In the comparison rna interference vector pSilencer5.1-Scramble liposome transfection A549 cell obtained above-mentioned two, puromycin (puromycin, 1.25 �� g/ml) screen viral infection positive cell clone, obtain A549/pSilencer5.1-Scramble.
4, westernblot identifies
DMEM culture medium containing 10% hyclone is cultivated A549/pLenti6/V5 compared with control cells (Con), A549/pLenti6/V5-HIP-55 cell (HIP-55), A549/pSilencer5.1-siRNA cell (HIP-55KD), A549/pSilencer5.1-Scramble cell (scr) and A549 cell (parental/par) 36 hours; Centrifugal collection supernatant; (Anti-HIP-55, BDBiosciences, catalog number: 612615) is detected with westernblot.
Result as depicted in figs. 1 and 2, Fig. 1 be RNA disturb HIP-55 cell in protein expression, Fig. 2 be process LAN HIP-55 cell in protein expression;
Fig. 1 all has in A549 cell (parental/par) and A549/pSilencer5.1-Scramble cell (scr) expression of destination protein HIP-55, and compared with A549/pSilencer5.1-Scramble cell (scr), RNA disturbs the expression of HIP-55 albumen in cell A549/pSilencer5.1-siRNA cell (HIP-55KD) of HIP-55 substantially to lower, and strikes and subtract efficiency more than 90%.
A549/pSilencer5.1-Scramble cell (scr) result and A549 cell (parental/par) are without significant difference.
In Fig. 2, A549/pLenti6/V5-HIP-55 cell (HIP-55) is compared with A549/pLenti6/V5 compared with control cells (Con), and the HIP-55 expressing quantity in A549/pLenti6/V5-HIP-55 cell (HIP-55) is apparently higher than A549/pLenti6/V5 compared with control cells (Con).
Adopt same method that empty carrier pSilencer5.1 is transfected A549 cell, obtain turning empty carrier cell A549/pSilencer5.1.
Embodiment 2, HIP-55 functional study
One, HIP-55 is to A549 cell proliferation and Apoptosis
1, the impact that lung cancer A549 cell is bred by HIP-55
Adopt the detection of Sulforhodamine (SRB) development process:
By A549 cell (parental/par), A549/pLenti6/V5(con) cell, the A549/pLenti6/V5-HIP-55 cell (HIP-55) obtained by embodiment 1, A549/pSilencer5.1-siRNA cell (HIP-55KD), A549/pSilencer5.1-Scramble cell (Scr) cultivate terminate after, take out culture plate, every hole adds 50%(mass/volume) the fixing cell of trichloroacetic acid (TCA) 50 �� L, final concentration of the 10% of TCA, gently it is added on the liquid level of every hole, in 4 DEG C of refrigerators, then places 1h. Culture plate each hole deionized water wash 5 times, to remove TCA. Dried in atmosphere, the SRB100LL that every hole adds 0.4%, ambient temperatare puts 10��30min, discard in each hole and wash 5 times with 1% acetic acid after liquid, remove unconjugated dyestuff, after air drying with pH be 10.5,10mmol/LunbufferedTrisbase (tri methylol amino methane) dissolve, vibrate 5min on oscillator plate, is that 490nm measures OD value at enzyme-linked immunosorbent assay instrument in wavelength. With A549/pSilencer5.1 cell and A549/pLenti6/V5(con) cell be comparison.
As shown in Figure 3 and Figure 4, the numerical value in figure is with first day OD value for 1 to result, and all the other are its ratio;
Fig. 3 is the RNA cell proliferation disturbing HIP-55, and wherein A549 cell (parental/par) cell is in the cell proliferation ratio respectively 1,1.402,2.218,2.569,3.661,5.703 cultivating 1,2,3,4,5,6 days;
A549/pSilencer5.1-Scramble cell (Scr) cell is in the cell proliferation ratio respectively 1,1.467,2.560,2.688,4.197,7.140 cultivating 1,2,3,4,5,6 days;
A549/pSilencer5.1-siRNA cell (HIP-55KD) is in the cell proliferation ratio respectively 1,1.052,1.579,1.983,2.705,3.05 cultivating 1,2,3,4,5,6 days;
Fig. 4 is the cell proliferation of process LAN HIP-55, wherein, A549/pLenti6/V5(con) cell cultivating the cell proliferation ratio of 1,2,3,4,5,6 days respectively 1,1.276,1.688,3.339,3.845,4.882;
A549/pLenti6/V5-HIP-55 cell (HIP-55) is in the cell proliferation ratio respectively 1,1.640,2.716,5.513,6.957,9.402 cultivating 1,2,3,4,5,6 days;
A549 cell and A549/pSilencer5.1 result are without significant difference.
It is shown that compare with A549 cell (parental, par) and A549/pSilencer5.1-Scramble cell (Scr), strike cell A549/pSilencer5.1-siRNA(HIP-55KD after subtracting HIP-55) propagation substantially weaken; In contrast, with A549/pLenti6/V5(con) compared with, after process LAN HIP-55, the propagation of cell A549/pLenti6/V5-HIP-55 cell (HIP-55) is remarkably reinforced.
Therefore strike and subtract HIP-55 suppression cell proliferation; Process LAN HIP-55 can improve cell proliferation.
2, the HIP-55 impact on lung cell A549 apoptosis
The A549/pSilencer5.1-siRNA cell (HIP-55KD) obtained by embodiment 1, A549/pSilencer5.1-Scramble cell (Scr) are inoculated in 12 orifice plates, complete medium is cultivated 24h, add tumor necrosis factor (tumornecrosisfactor-��, TNF-��; BD company, article No.: 354066) apoptosis-induced (100ng/ml), collect cell at different time and wash three times with PBS, 7-AAD and Annexin5-PE dyes, mixing, room temperature lucifuge reaction 15min, flow cytometer (Guavaflowcytometersystems, Millipore) detects apoptosis rate.
Result as it is shown in figure 5,
A549/pSilencer5.1-Scramble cell (Scr) 0,6, the apoptosis rate of 9h respectively 7.2%, 11.3%, 29.3%;
A549/pSilencer5.1-siRNA(HIP-55KD) 0,6, the apoptosis rate of 9h respectively 12.8%, 30%, 54.8%;
Application westernblot detects apoptosis markers CleavedCaspase-3(and sells company cellsignaling; Catalog number #9664) and Parp(sale company cellsignaling; Catalog number #9532), result as shown in Figure 6, is struck after subtracting HIP-55 in A549 cell, and caspase3 and the Parp of shear pattern substantially increases, show that apoptosis substantially increases, after A549 cell process LAN HIP-55, then reverse this biological process completely.
The above results shows, strikes after subtracting HIP-55 in A549 cell, it is possible to promote apoptosis, improves apoptosis rate.
Two, the HIP-55 impact on Cell clonality
Adopting soft-agar cloning to be formed to analyze, the agarose gel using 1% is as deposit glue, and when being cooled to 50 DEG C, the bottom glue being mixed in proportion preparation 0.6% with culture medium is laid in culture dish;When being cooled to 42 DEG C, mix with cell suspension in proportion, the top-layer agar of 0.2%. after top-layer agar solidifies, cover lid layer complete medium to prevent agar surface from drying on its surface, it is then placed in cell culture incubator and cultivates, every day Microscopic observation, and supplementing culture medium, cell clone group Colony forming under light microscopic after 2 weeks.
Above-mentioned cell suspension is that cell is cultivated suspension that 2 time-of-weeks obtain in the medium.
Above-mentioned cell is A549 cell (parental/par), A549/pSilencer5.1-siRNA cell (HIP-55KD), A549/pSilencer5.1-Scramble cell (Scr). With A549/pSilencer5.1 for comparison.
Shown in result such as Fig. 7 (wherein left figure is result of taking pictures, and right figure is clone's number statistical result),
The number of cell clones of A549 cell (parental/par) is 76.6;
The number of cell clones of A549/pSilencer5.1-Scramble cell (Scr) is 105
The number of cell clones of A549/pSilencer5.1-siRNA cell (HIP-55KD) is 38;
A549 cell (parental/par) and A549/pSilencer5.1 result are without significant difference.
The above results shows, strikes after subtracting HIP-55 in A549 cell, compares with A549 cell (parental, par) and A549/pSilencer5.1-Scramble cell (Scr), and Cell clonality substantially reduces.
Three, HIP-55 on cell migration impact
1) scratch test
Spreading in 6 orifice plates into cell, after cell fusion, with the 200 aseptic liquid transfer gun head cuts of �� L, PBS carefully washes away floating cells to be repeated 3 times, basis of microscopic observation after cultivation 24h.
Above-mentioned cell is A549 cell (parental/par), A549/pSilencer5.1-siRNA cell (HIP-55KD), A549/pSilencer5.1-Scramble cell (Scr). With A549/pSilencer5.1 for comparison.
Result is as shown in Figure 8, it can be seen that strike after subtracting HIP-55 in A549 cell, compares with A549 cell (parental/par) and siRNA unrelated sequences compared with control cells (scramble, scr), and cell migration ability substantially reduces.
A549 cell (parental/par) and A549/pSilencer5.1 result are without significant difference.
2) Transwell Cell migration assay
Transwell (8 ��m of poresize; The every hole of BDBiosciences cell adds 1 �� 105Individual cell, under cell, room adds the DMEM containing hyclone, after 37 DEG C of cultivation 24h, cell that is that wipe microporous membrane upper strata with cotton swab and that do not attack people's basement membrane, the cell methanol of invasion and attack to subsurface is fixed, haematoxylin dyeing. Under inverted microscope, counting moves to the cell of microporous membrane lower floor, inserts 750mL/L alcohol fixation 15min, violet staining 15min, and PBS rinses repeatedly, washes away unnecessary crystal violet. Observe under 200 power microscopes, 5 visuals field of random counter, take average.
Above-mentioned cell is A549 cell (parental/par), A549/pSilencer5.1-siRNA cell (HIP-55KD), A549/pSilencer5.1-Scramble cell (Scr), A549/pLenti6/V5 cell (Con), A549/pLenti6/V5-HIP-55 cell (HIP-55); With A549/pSilencer5.1 for comparison.
Result is as it is shown in figure 9, A is interference result, and B is process LAN result, and wherein left figure is microscope and takes pictures result, and right figure is cartogram; It can be seen that
A549(parental/par) cell quantity through Transwell cell film is 281;
A549/pSilencer5.1-siRNA(HIP-55KD) cell quantity through Transwell cell film is 103;
A549/pSilencer5.1-Scramble(Scr) cell quantity through Transwell cell film is 306.
A549/pLenti6/V5 compared with control cells (Con) is 220 through the cell quantity of Transwell cell film;
A549/pLenti6/V5-HIP-55 cell (HIP-55) is 468 through the cell quantity of Transwell cell film.
It can be seen that after striking in A549 cell and subtracting HIP-55, compare with A549 cell (parental/par) and siRNA unrelated sequences compared with control cells (scramble, scr), cell migration ability substantially reduces.
In A549 cell after process LAN HIP-55, compared with A549 cell (Con), cell migration ability substantially increases.
Four, cell invasion is affected by HIP-55
Transwell is coated Matrigel glue bottom upper room in advance, adds the culture medium aquation of a small amount of serum-free of people before using. All the other migrate experiment with Transwell cell.
In interference group, cell is A549 cell (parental/par), A549/pSilencer5.1-siRNA cell (HIP-55KD), A549/pSilencer5.1-Scramble cell (Scr);
In process LAN group, cell is A549/pLenti6/V5 compared with control cells (Con), A549/pLenti6/V5-HIP-55 cell (HIP-55).
As shown in Figure 10, A is interference result to result, and B is process LAN result, and wherein left figure is microscope and takes pictures result, and right figure is cartogram;
It is 471 that A549 cell (parental/par) attacks the cell quantity of Matrigel traverse Transwell cell film;
It is 150 that A549/pSilencer5.1-siRNA cell (HIP-55KD) attacks the cell quantity of Matrigel traverse Transwell cell film;
It is 565 that A549/pSilencer5.1-Scramble cell (Scr) attacks the cell quantity of Matrigel traverse Transwell cell film.
Showing, strike after subtracting HIP-55 in A549 cell, as compared to A549 cell (parental, par) and siRNA unrelated sequences compared with control cells (scramble, scr), cell invasion ability substantially reduces.
It is 84 that A549/pLenti6/V5 compared with control cells (Con) attacks the cell quantity of Matrigel traverse Transwell cell film;
It is 238 that A549/pLenti6/V5-HIP-55 cell (HIP-55) attacks the cell quantity of Matrigel traverse Transwell cell film.
Showing, in A549 cell after process LAN HIP-55, compared with A549/pLenti6/V5 compared with control cells (Con), cell invasion ability substantially increases.
Five, the HIP-55 impact on becoming tumor
The cell of In vitro culture, collects cell and adjustment is resuspended in PBS to suitable concentration, takes 200 �� l cell suspension (2 �� 106Cell) in Balb/c nude mice (4-5 week) oxter subcutaneous injection. Raising nude mice 4 weeks, naked eyes are obvious tumor body as seen, takes tumor and measures tumor weight and take pictures.
Above-mentioned cell is A549 cell (parental/par), A549/pSilencer5.1-siRNA cell (HIP-55KD), A549/pLenti6/V5-HIP-55 cell (HIP-55/WT), A549/pSilencer5.1-Scramble cell (Scr).
As shown in figure 11, left figure is detection HIP-55 Westernblot figure, the right figure expressed is statistics tumor weight figure to result;
After AA549/pLenti6/V5-HIP-55 cell (HIP-55/WT) injection, tumor weight is 1.418g;
After A549/pSilencer5.1-siRNA cell (HIP-55KD) injection, tumor weight is 0.253g;
After A549/pSilencer5.1-Scramble cell (Scr) injection, tumor weight is 0.92g;
A549 cell (parental/par) and A549/pSilencer5.1-Scramble cell (Scr) result are without significant difference.
It is shown that strike in A549 cell subtract HIP-55 after nude mouse tumor weight substantially reduce, and after process LAN HIP-55, tumor weight is significantly raised.
Six, HIP-55 high expressed in patients with lung cancer tissue samples
For the clear and definite HIP-55 expression at patients with lung cancer, application immunohistochemical method have detected normal lung (make a definite diagnosis, and patient knows the inside story) tissue and the expression of HIP-55 in patients with lung cancer (make a definite diagnosis, and patient knows the inside story) lung tissue.
As shown in figure 12, upper figure is ImmunohistochemistryResults Results to result, and figure below is cartogram; Result shows compared with normal lung tissue HIP-55 extremely substantially high expressed (Figure 12 figure below) in Lung Tissue.
Meanwhile, application prepare the polypeptide of HIP-55 antibody and is closed after cancerous lung tissue sample the clear and definite gained positive findings of method of Immunohistochemical detection HIP-55 expression whether from unspecific staining. It is shown that the polypeptide preparing HIP-55 antibody can block the dyeing to cancerous lung tissue of HIP 55 antibody after closing cancerous lung tissue sample completely, illustrate that positive staining is HIP 55 antibody specificity (the upper figure of Figure 12).
Claims (4)
1. a recombinant vector, for the DNA molecular of coded interference HIP-55 protein expression siRNA is inserted expression vector, obtains recombinant vector; The aminoacid sequence of described HIP-55 albumen is the sequence 2 in sequence table; The nucleotides sequence of the DNA molecular of described coded interference HIP-55 protein expression siRNA is classified as in sequence table sequence 5.
2. the recombinant vector described in claim 1 has following 1 in preparation)-7) at least one function product in application:
1) cell proliferation is suppressed;
2) apoptosis is promoted;
3) tumor cell migration is suppressed;
4) tumor cell invasion is suppressed;
5) tumor cell clone is suppressed to be formed;
6) tumor is suppressed to be formed;
7) prevention and/or treatment tumor;
Described tumor is pulmonary carcinoma;
Described tumor cell is tumor cell; Described tumor cell is lung carcinoma cell.
3. application according to claim 2, it is characterised in that: described product is medicine.
4. the application according to Claims 2 or 3, it is characterised in that: described lung carcinoma cell behaviour non-small cell lung cancer cell.
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