CN103773802A - Application of HIP-55 protein to development of tumor inhibition drugs - Google Patents
Application of HIP-55 protein to development of tumor inhibition drugs Download PDFInfo
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Abstract
The invention discloses application of HIP-55 protein to development of tumor inhibition drugs. DNA molecules encoding and interfering HIP-55 protein from expressing siRNA are inserted into an expression vector to obtain a recombinant vector; the amino acid sequence of the HIP-55 protein is a sequence II in a sequence table; the invention also provides application of HIP-55 protein or the recombinant vector containing the HIP-55 protein coding gene to preparation of products having the following functions of (1) promoting tumor cell migration and/or (2) promoting tumor cell invasion. Experiments prove that the HIP-55 protein gene is overexpressed in lung cancer cells to promote tumor formation, lung cancer cell migration and lung cancer cell invasion; therefore, the HIP-55 provides a new target for clinical diagnosis, treatment and development of new drugs and can be used for screening, developing and/or designing products for prevention and/or treatment of tumors.
Description
Technical field
The present invention relates to biological technical field, relate in particular to a kind of HIP-55 albumen and suppress the application in tumour medicine in exploitation.
Background technology
HIP-55 (hematopoietic progenitor kinase 1[HPK1]-interacting protein of55kDa; Also referred to as drebrin-like protein DBNL, mAbp1 or SH3P7) be a protein that comprises multi-functional territory.Its structure is mainly comprising F-actin protein binding structural domain and the C end SH3 structural domain of N end.HIP-55 participates in cynapse generation, acceptor endocytosis, cytoskeleton rearrangement and signal transduction.At present HIP-55 function is understood lessly, known HIP-55 plays an important role in immunity system by regulating φt cell receptor endocytosis, release of cytokines and B-cell receptor to present etc.Research recently shows that HIP-55 is by interacting and regulate cytoskeleton rearrangement in neural system with N-WASP and Arp2/3.But its effect in tumour all there is no research.
Summary of the invention
An object of the present invention is to provide a kind of recombinant vectors.
Recombinant vectors provided by the invention, for the DNA molecular of coded interference HIP-55 protein expression siRNA is inserted to expression vector, obtains recombinant vectors; The aminoacid sequence of described HIP-55 albumen is the sequence 2 in sequence table.
In above-mentioned recombinant vectors, the nucleotides sequence of the DNA molecular of described coded interference HIP-55 protein expression siRNA is classified sequence 5 in sequence table as.In an embodiment of the present invention, described expression vector is pSilencer5.1; Recombinant vectors is pSilencer5.1-siRNA, for the DNA molecular (sequence 5) of coding siRNA is inserted between the BamHI and HindIII restriction enzyme site of pSilencer5.1 carrier, obtains the carrier of the HIP-55siRNA shown in expressed sequence 3.
Above-mentioned recombinant vectors has following 1 in preparation)-7) in application in the product of at least one function be also the scope of protection of the invention:
1) suppress cell proliferation;
2) promote apoptosis;
3) inhibition tumor cell migration;
4) inhibition tumor cell invasion and attack;
5) inhibition tumor cell clone forms;
6) suppressing tumour forms;
7) prevent and/or treat tumour.
In above-mentioned application, described product is medicine.
In above-mentioned application, described tumour is lung cancer; Described tumour cell is tumour cell.
In above-mentioned application, described tumour cell is lung carcinoma cell; Described lung carcinoma cell is specially Non-small cell lung carcinoma cell.
The application of the recombinant vectors that another object of the present invention is to provide HIP-55 albumen or its encoding gene or contains HIP-55 protein coding gene.
HIP-55 albumen provided by the invention or its encoding gene or the recombinant vectors that contains HIP-55 protein coding gene have following 1 in preparation) and/or 2) application in the product of function:
1) promote tumor cell migration;
2) promote tumor cell invasion;
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 the sequence 1 in sequence table as.
In above-mentioned application, described product is medicine.
In above-mentioned application, described tumour is lung cancer;
Described tumour cell is tumour cell; Described tumour cell is lung carcinoma cell; In an embodiment of the present invention, described lung carcinoma cell behaviour non-small cell lung cancer cell, is specially Non-small cell lung carcinoma cell A549.
The application that HIP-55 albumen prevents and/or treats in tumour product in screening, exploitation and/or design is also the scope of protection of the invention; The aminoacid sequence of described HIP-55 albumen is the sequence 2 in sequence table.
Of the present invention experimental results show that, the present invention finds HIP-55 obvious high expression level in clinical lung cancer patient tissue samples, further research shows the function of HIP-55: by cross the gene of expressing this albumen in lung carcinoma cell, can promote tumour to form, promote lung carcinoma cell migration and lung carcinoma cell invasion; Disturb reticent HIP-55 albumen by RNA, can obviously suppress tumour and form, promote apoptosis, suppress propagation, cell clonal formation, cell migration and/or cell invasion, can be used to preparation and prevent and/or treat tumour product.Therefore, HIP-55 is that clinical diagnosis, treatment and new drug development have been carried for new target spot, can be for preventing and/or treating tumour product in screening, exploitation and/or design.
Accompanying drawing explanation
Fig. 1 is that RNA disturbs protein expression in the cell of HIP-55
Fig. 2 expresses protein expression in the cell of HIP-55
Fig. 3 is the cell proliferation that RNA disturbs HIP-55
Fig. 4 was the cell proliferation of expressing HIP-55
Fig. 5 is the impact of HIP-55 on lung cell A549 apoptosis
Fig. 6 is the impact of HIP-55 on apoptosis marker
Fig. 7 is the impact of HIP-55 on Cell clonality
Fig. 8 is the impact that scratch test detects HIP-55 on cell migration
Fig. 9 is the impact that Transwell detects HIP-55 on cell migration
Figure 10 is that HIP-55 affects cell invasion
Figure 11 is that HIP-55 is on becoming the impact of knurl
Figure 12 is HIP-55 high expression level in patients with lung cancer tissue samples
Embodiment
The experimental technique using in following embodiment if no special instructions, is ordinary method.
Material, reagent etc. used in following embodiment, if no special instructions, all can obtain from commercial channels.
In following embodiment quantitative experiment all in triplicate, results averaged.
In following embodiment, Anti-HIP-55 antibody is purchased from BD Bioscience (San Diego, CA).
Non-small cell lung carcinoma A549(U.S. ATCC in following embodiment, catalog number CCL-185
tM) in the nutrient solution of the DMEM in high glucose that contains 10% foetal calf serum, 100U/ml penicillin, 100U/ml Streptomycin sulphate, cultivate, and be placed in 37 ℃, the CO2gas incubator of 5% concentration.A549 cell is through had digestive transfer culture, and the vegetative period cell of taking the logarithm is stand-by.
The aminoacid sequence of people HIP-55 albumen is the sequence 2 in sequence table, and the nucleotides sequence of the gene of this albumen of encoding is classified the sequence 1 in sequence table as.
One, the acquisition of HIP-55siRNA
Design and synthesize following siRNA sequence for people HIP-55 protein coding gene:
HIP-55 siRNA, its nucleotides sequence is classified 5'-GAUCCG CAGUGAACGUAGAGAAUUG UUCAAGAGACAAUUCUCUACGUUCACUGUU UUUUGGAAA-3'(sequence 3 as);
Contrast Scramble siRNA, its nucleotides sequence is classified 5'-GAUCCG CACUACCGGUUGUAUAGGUGUUCAAGAGA CACCUAUACAAAGGUAGUG UUUUGGAAA-3'(sequence 4 as).
Two, cross the structure of expressing the recombinant vectors of HIP-55 and the recombinant vectors of RNA interference HIP-55
1, cross the recombinant vectors pLenti6/V5-HIP-55 structure of expressing HIP-55
Take the gene of the HIP-55 shown in sequence 1 in above-mentioned sequence table as template, use
Forward: '-GGGGACAAGTTTGTACAAAAAAGCAGGCTTC ATGGCGGCGAACCTGAGCC-3 ' and
Reverse:5 '-GGGGACCACTTTGTACAAGAAAGCTGGGTCCTCAATGAGCTCCACGTAGT-3 ' primer increases, and obtains the PCR product of 1356bp.
By the PCR product of 1356bp and pDONR221 carrier (purchased from Invitrogen, catalog number is 12536017) carry out BP reaction, obtain recombinant vectors 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 vectors pLenti6/V5-HIP-55.Through order-checking, the DNA molecular shown in the sequence in sequence table 1 is inserted the carrier obtaining between the pLenti6/V5-DEST of expression vector by this recombinant vectors pLenti6/V5-HIP-55.
2, RNA disturbs the structure of the recombinant vectors pSilencer5.1-siRNA of HIP-55
The DNA molecular of composite coding HIP-55 siRNA, its nucleotides sequence is classified as; 5'-GATCCGCAGTGAACGTAGAGAATTG TTCAAGAGA CAATTCTCTACGTTCACTGTT TTTTGGAAA-3'(sequence 5); Formed by the annealing of two single stranded DNAs, the nucleotides sequence that wherein nucleotides sequence of a single stranded DNA is classified another single stranded DNA of F:5'-GATCCGCAGTGAACGTAGAGAATTG TTCAAGAGA CAATTCTCTACGTTCACTGTT TTTTGGAAA-3' as is classified R:5'-AGCTTTTCCAAAA AACAGTGAACGTAGAGAATTG TCTCTTGAACAATTCTCTACGTTCACTG CG-3' as.
The DNA molecular (sequence 5) of coding HIP-55 siRNA is inserted pSilencer5.1(Invitrogen by rna interference vector pSilencer5.1-siRNA, AM5782), between the BamHI of carrier and HindIII restriction enzyme site, obtain the carrier of the HIP-55 siRNA shown in expressed sequence 3.
This DNA molecular is made up of forward DNA fragmentation, stuffer fragment and reverse 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 reverse DNA fragment be in sequence table sequence 5 from the DNA molecular shown in 5 ' end 35-53 position Nucleotide.
Contrast rna interference vector pSilencer5.1-Scramble: for the DNA molecular of coding Scramble siRNA is inserted between the BamHI and HindIII site of pSilencer5.1 carrier, obtain the carrier of the Scramble siRNA shown in expressed sequence 4.
The DNA molecular of above-mentioned coding Scramble siRNA, its nucleotides sequence is classified 5'-GATCCGCACTACCGGTTGTATAGGTG TTCAAGAGA CACCTATACAAAGGTAGTG TTTTGGAAA-3'(sequence 6 as); This DNA molecular is made up of forward DNA fragmentation, stuffer fragment and reverse 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 reverse DNA fragment be in sequence table sequence 6 from 5 ' end 35-53 position Nucleotide.
Three, cross the structure of expressing the A549 cell of HIP-55 and the A549 cell of RNA interference HIP-55
1, cross the structure of the A549 cell of expressing HIP-55
The above-mentioned two recombinant vectors pLenti6/V5-HIP-55 that obtain are infected in A549 cell, obtain A549/pLenti6/V5-HIP-55.
2, contrasted the structure of the A549 cell of expressing HIP-55
Expression vector pLenti6/V5-DEST is infected in A549 cell, obtain A549/pLenti6/V5.
3, RNA disturbs the structure of the A549 cell of HIP-55
By in the above-mentioned two rna interference vector pSilencer5.1-siRNA liposome transfection A549 cells that obtain, tetracycline (puromycin, 1.25 μ g/ml) screening virus infection positive cell clone, obtains A549/pSilencer5.1-siRNA.
4, contrast RNA disturbs the structure of A549 cell
By in the above-mentioned two contrast rna interference vector pSilencer5.1-Scramble liposome transfection A549 cells that obtain, tetracycline (puromycin, 1.25 μ g/ml) screening virus infection positive cell clone, obtain A549/pSilencer5.1-Scramble.
4, western blot identifies
Containing cultivating 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) in the DMEM substratum of 10% foetal calf serum 36 hours; Centrifugal collection supernatant liquor; By western blot detection (Anti-HIP-55, BD Biosciences, catalog number: 612615).
As depicted in figs. 1 and 2, Fig. 1 is that RNA disturbs protein expression in the cell of HIP-55 to result, and Fig. 2 expresses protein expression in the cell of HIP-55;
In Fig. 1, in A549 cell (parental/par) and A549/pSilencer5.1-Scramble cell (scr), all there is the expression of target protein HIP-55, and compared with A549/pSilencer5.1-Scramble cell (scr), RNA disturbs the expression amount of HIP-55 albumen in the cell A549/pSilencer5.1-siRNA cell (HIP-55KD) of HIP-55 obviously 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 and use the same method empty carrier pSilencer5.1 transfection A549 cell, obtain turning empty carrier cell A549/pSilencer5.1.
The functional study of embodiment 2, HIP-55
One, HIP-55 is to A549 cell proliferation and Apoptosis
1, the impact of HIP-55 on lung cancer A549 cell propagation
Adopt sulphonyl rhodamine (SRB) development process to detect:
By A549 cell (parental/par), A549/pLenti6/V5(con) after cell, the A549/pLenti6/V5-HIP-55 cell (HIP-55) being obtained by embodiment 1, A549/pSilencer5.1-siRNA cell (HIP-55KD), A549/pSilencer5.1-Scramble cell (Scr) cultivate and finish, take out culture plate, every hole adds 50%(mass/volume) trichoroacetic acid(TCA) (TCA) 50 μ L fixed cells, the final concentration of TCA is 10%, gently be added on the liquid level of every hole, then in 4 ℃ of refrigerators, place 1h.Deionized water wash 5 times of the each hole of culture plate, to remove TCA.After air drying, every hole adds 0.4% SRB100LL, under room temperature, place 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/L unbuffered Tris base (tri methylol amino methane) dissolves, the 5min that vibrates on oscillator plate, is that 490nm measures OD value at enzyme-linked immunosorbent assay instrument in wavelength.Take A549/pSilencer5.1 cell and A549/pLenti6/V5(con) cell be contrast.
As shown in Figure 3 and Figure 4, the numerical value in figure is take first day OD value as 1 to result, and all the other are its ratio;
Fig. 3 is the cell proliferation that RNA disturbs HIP-55, and wherein A549 cell (parental/par) cell is respectively 1,1.402,2.218,2.569,3.661,5.703 in the cell proliferation ratio of cultivating 1,2,3,4,5,6 day;
A549/pSilencer5.1-Scramble cell (Scr) cell is respectively 1,1.467,2.560,2.688,4.197,7.140 in the cell proliferation ratio of cultivating 1,2,3,4,5,6 day;
A549/pSilencer5.1-siRNA cell (HIP-55KD) is respectively 1,1.052,1.579,1.983,2.705,3.05 in the cell proliferation ratio of cultivating 1,2,3,4,5,6 day;
Fig. 4 expresses the cell proliferation of HIP-55, wherein, A549/pLenti6/V5(con) cell is respectively 1,1.276,1.688,3.339,3.845,4.882 in the cell proliferation ratio of cultivating 1,2,3,4,5,6 day;
A549/pLenti6/V5-HIP-55 cell (HIP-55) is respectively 1,1.640,2.716,5.513,6.957,9.402 in the cell proliferation ratio of cultivating 1,2,3,4,5,6 day;
A549 cell and A549/pSilencer5.1 result are without significant difference.
Result shows, compares with A549 cell (parental, par) and A549/pSilencer5.1-Scramble cell (Scr), strikes and subtracts cell A549/pSilencer5.1-siRNA(HIP-55KD after HIP-55) propagation obviously weaken; In contrast, with A549/pLenti6/V5(con) compared with, after expression HIP-55, the propagation of cell A549/pLenti6/V5-HIP-55 cell (HIP-55) obviously strengthens excessively.
Therefore strike and subtract HIP-55 inhibition cell proliferation; Cross expression HIP-55 and can improve cell proliferation.
2, the impact of HIP-55 on lung cell A549 apoptosis
The A549/pSilencer5.1-siRNA cell (HIP-55KD) being obtained by embodiment 1, A549/pSilencer5.1-Scramble cell (Scr) are inoculated in to 12 orifice plates, in perfect medium, cultivate 24h, add tumour necrosis factor (tumor necrosis factor-α, TNF-α; BD company, article No.: 354066) apoptosis-induced (100ng/ml), wash three times at different time collecting cell and with PBS, 7-AAD and Annexin5-PE dyeing, mix, room temperature lucifuge reaction 15min, flow cytometer (Guava flow cytometer systems, Millipore) detects apoptosis rate.
Result as shown in Figure 5,
A549/pSilencer5.1-Scramble cell (Scr) 0,6, the apoptosis rate of 9h is respectively 7.2%, 11.3%, 29.3%;
A549/pSilencer5.1-siRNA(HIP-55KD) 0,6, the apoptosis rate of 9h is respectively 12.8%, 30%, 54.8%;
Application western blot detects apoptosis marker Cleaved Caspase-3(and sells the cellsignaling of company; Catalog number #9664) and the Parp(sale cellsignaling of company; Catalog number #9532), result as shown in Figure 6, is struck and is subtracted after HIP-55 in A549 cell, and caspase 3 and the Parp of shear-form obviously increase, show that apoptosis obviously increases, after A549 cell is crossed expression HIP-55, reverse this biological procedures completely.
The above results shows, in A549 cell, strikes and subtracts after HIP-55, can promote apoptosis, improves apoptosis rate.
Two, the impact of HIP-55 on Cell clonality
Adopt soft-agar cloning to form and analyze, the sepharose using 1% is as deposit glue, and while being cooled to 50 ℃, the bottom glue that is mixed in proportion preparation 0.6% with substratum is laid in culture dish; While being cooled to again 42 ℃, mix with cell suspension in proportion, 0.2% top-layer agar. dry to prevent agar surface at its surface coverage one deck perfect medium after top-layer agar solidifies, then putting into cell culture incubator cultivates, every day Microscopic observation, and supplemental medium, after 2 weeks, under light microscopic, cell clone group colony forms.
Above-mentioned cell suspension is cell to be cultivated in substratum to the suspension that 2 time-of-weeks obtain.
Above-mentioned cell is A549 cell (parental/par), A549/pSilencer5.1-siRNA cell (HIP-55KD), A549/pSilencer5.1-Scramble cell (Scr).Take A549/pSilencer5.1 as contrast.
Result shown in Fig. 7 (wherein left figure is the result of taking pictures, and right figure is clone's number statistics),
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, in A549 cell, strikes and subtracts after HIP-55, compares with A549 cell (parental, par) and A549/pSilencer5.1-Scramble cell (Scr), and Cell clonality obviously reduces.
Three, HIP-55 on cell migration impact
1) scratch test
In 6 orifice plates, spread into cell, after cytogamy, with the aseptic liquid transfer gun head cut of 200 μ L, PBS carefully washes away floating cell and repeats 3 times, micro-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).Take A549/pSilencer5.1 as contrast.
Result as shown in Figure 8, can be found out, in A549 cell, strikes and subtracts after HIP-55, compares with A549 cell (parental/par) and the irrelevant sequence compared with control cells (scramble, scr) of siRNA, and cell migration ability obviously reduces.
A549 cell (parental/par) and A549/pSilencer5.1 result are without significant difference.
2) Transwell cell migration experiment
Transwell (8 μ mporesize; The every hole of BD Biosciences cell adds 1 × 10
5individual cell, under cell, chamber adds the DMEM containing foetal calf serum, cultivates after 24h for 37 ℃, wipes cell microporous membrane upper strata and that do not attack people's basilar membrane with cotton swab, the cell methyl alcohol of attacking to subsurface is fixed to 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 is rinsing repeatedly, washes away unnecessary Viola crystallina.Under 200 power microscopes, observe, count at random 5 visuals field, get 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); Take A549/pSilencer5.1 as contrast.
As shown in Figure 9, A is for disturbing result for result, and B was expression of results, and wherein left figure is the microscope result of taking pictures, and right figure is statistical graph; Can find out,
A549(parental/par) be 281 through the cell quantity of Transwell cell film;
A549/pSilencer5.1-siRNA(HIP-55KD) be 103 through the cell quantity of Transwell cell film;
A549/pSilencer5.1-Scramble(Scr) be 306 through the cell quantity of Transwell cell film.
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.
Can find out, in A549 cell, strike and subtract after HIP-55, compare with A549 cell (parental/par) and the irrelevant sequence compared with control cells (scramble, scr) of siRNA, cell migration ability obviously reduces.
In A549 cell, cross after expression HIP-55, compared with A549 cell (Con), cell migration ability obviously increases.
Four, HIP-55 affects cell invasion
Bottom, the upper chamber of Transwell is coated with Matrigel glue in advance, adds the substratum aquation of a small amount of serum-free of people before use.All the other are with Transwell cell migration experiment.
In interference group, cell is A549 cell (parental/par), A549/pSilencer5.1-siRNA cell (HIP-55KD), A549/pSilencer5.1-Scramble cell (Scr);
Crossing cell in expression group is A549/pLenti6/V5 compared with control cells (Con), A549/pLenti6/V5-HIP-55 cell (HIP-55).
As shown in figure 10, A is for disturbing result for result, and B was expression of results, and wherein left figure is the microscope result of taking pictures, and right figure is statistical graph;
A549 cell (parental/par) invasion and attack Matrigel is 471 through the cell quantity of Transwell cell film;
A549/pSilencer5.1-siRNA cell (HIP-55KD) invasion and attack Matrigel is 150 through the cell quantity of Transwell cell film;
A549/pSilencer5.1-Scramble cell (Scr) invasion and attack Matrigel is 565 through the cell quantity of Transwell cell film.
Show, in A549 cell, strike and subtract after HIP-55, compare with A549 cell (parental, par) and the irrelevant sequence compared with control cells (scramble, scr) of siRNA, cell invasion ability obviously reduces.
A549/pLenti6/V5 compared with control cells (Con) invasion and attack Matrigel is 84 through the cell quantity of Transwell cell film;
A549/pLenti6/V5-HIP-55 cell (HIP-55) invasion and attack Matrigel is 238 through the cell quantity of Transwell cell film.
Show, in A549 cell, cross after expression HIP-55, compared with A549/pLenti6/V5 compared with control cells (Con), cell invasion ability obviously increases.
Five, HIP-55 is on becoming the impact of knurl
The cell of vitro culture, collecting cell is also adjusted to suitable concentration and is resuspended in PBS, gets 200 μ l cell suspensions (2 × 10
6cell) in Balb/c nude mice (4-5 week) oxter subcutaneous injection.Raise nude mice 4 weeks, the visible obviously knurl body of naked eyes, gets knurl 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 detects the Western blot figure that HIP-55 expresses to result, and right figure is statistics tumor weight figure;
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.
Result shows, in A549 cell, strike subtract HIP-55 after nude mice tumor weight obviously reduce, and after crossing expression HIP-55, tumor weight obviously raises.
Six, HIP-55 high expression level in patients with lung cancer tissue samples
For clear and definite HIP-55 is at the expression of patients with lung cancer, application immunohistochemical method has detected the expression of HIP-55 in normal lung (make a definite diagnosis, and patient knowing the inside story) tissue and patients with lung cancer (make a definite diagnosis, and patient knowing the inside story) lung tissue.
As shown in figure 12, upper figure is ImmunohistochemistryResults Results to result, and figure below is statistical graph; Result shows compared with normal lung tissue extremely obviously high expression level (Figure 12 figure below) of HIP-55 in Lung Tissue.
Whether the clear and definite gained positive findings of method that meanwhile, after the polypeptide sealing cancerous lung tissue sample of application preparation HIP-55 antibody, immunohistochemistry detects HIP-55 expression is from unspecific staining.Result shows, after the polypeptide sealing cancerous lung tissue sample of preparation HIP-55 antibody, can block the dyeing of HIP-55 antibody to cancerous lung tissue completely, illustrates that positive staining is HIP-55 antibodies specific (the upper figure of Figure 12).
Claims (10)
1. a recombinant vectors, for the DNA molecular of coded interference HIP-55 protein expression siRNA is inserted to expression vector, obtains recombinant vectors; The aminoacid sequence of described HIP-55 albumen is the sequence 2 in sequence table.
2. recombinant vectors according to claim 1, is characterized in that: the nucleotides sequence of the DNA molecular of described coded interference HIP-55 protein expression siRNA is classified sequence 5 in sequence table as.
3. the recombinant vectors described in claim 1 or 2 has following 1 in preparation)-7) in application in the product of at least one function:
1) suppress cell proliferation;
2) promote apoptosis;
3) inhibition tumor cell migration;
4) inhibition tumor cell invasion and attack;
5) inhibition tumor cell clone forms;
6) suppressing tumour forms;
7) prevent and/or treat tumour.
4. application according to claim 3, is characterized in that: described product is medicine.
5. according to the application described in claim 3 or 4, it is characterized in that: described tumour is lung cancer;
Described tumour cell is tumour cell.
6. application according to claim 5, is characterized in that: described tumour cell is lung carcinoma cell; Described lung carcinoma cell is specially Non-small cell lung carcinoma cell.
7.HIP-55 albumen or its encoding gene or the recombinant vectors that contains HIP-55 protein coding gene have following 1 in preparation) and/or 2) application in the product of function:
1) promote tumor cell migration;
2) promote tumor cell invasion;
The aminoacid sequence of described HIP-55 albumen is the sequence 2 in sequence table.
8. application according to claim 7, is characterized in that:
The nucleotides sequence of described HIP-55 protein coding gene is classified the sequence 1 in sequence table as.
9. according to the application described in claim 7 or 8, it is characterized in that:
Described product is medicine;
Described tumour is lung cancer;
Described tumour cell is tumour cell; Described tumour cell is specially lung carcinoma cell;
Described lung carcinoma cell is especially specially Non-small cell lung carcinoma cell.
10.HIP-55 albumen prevents and/or treats the application in tumour product in screening, exploitation and/or design; The aminoacid sequence of described HIP-55 albumen is the sequence 2 in sequence table.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410045873.4A CN103773802B (en) | 2014-02-08 | 2014-02-08 | HIP-55 albumen suppresses the application in tumour medicine in exploitation |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN201410045873.4A CN103773802B (en) | 2014-02-08 | 2014-02-08 | HIP-55 albumen suppresses the application in tumour medicine in exploitation |
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| CN111965367A (en) * | 2020-07-23 | 2020-11-20 | 北京大学第三医院（北京大学第三临床医学院） | Application of HIP-55 as GPCR biased drug target |
| CN111991550A (en) * | 2020-08-07 | 2020-11-27 | 北京大学第三医院（北京大学第三临床医学院） | Application of HIP-55 in preparation of products for diagnosing and preventing myocardial ischemia-reperfusion injury |
| CN112480249A (en) * | 2020-11-26 | 2021-03-12 | 北京大学第三医院（北京大学第三临床医学院） | Preparation method and application of phosphorylated antibody product of AKT new substrate HIP-55 |
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| CN106086171A (en) * | 2016-06-15 | 2016-11-09 | 广州中大南沙科技创新产业园有限公司 | A kind of drug screening method suppressing tumor cell proliferation |
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| CN111965367A (en) * | 2020-07-23 | 2020-11-20 | 北京大学第三医院（北京大学第三临床医学院） | Application of HIP-55 as GPCR biased drug target |
| CN111991550A (en) * | 2020-08-07 | 2020-11-27 | 北京大学第三医院（北京大学第三临床医学院） | Application of HIP-55 in preparation of products for diagnosing and preventing myocardial ischemia-reperfusion injury |
| CN111991550B (en) * | 2020-08-07 | 2022-11-01 | 北京大学第三医院（北京大学第三临床医学院） | Application of HIP-55 in preparation of products for diagnosing and preventing myocardial ischemia reperfusion injury |
| CN112480249A (en) * | 2020-11-26 | 2021-03-12 | 北京大学第三医院（北京大学第三临床医学院） | Preparation method and application of phosphorylated antibody product of AKT new substrate HIP-55 |
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