CN102352356B - Bcl11a siRNA-2292 restraining expression of BCL11A and proliferation of tumorous B cells and application thereof - Google Patents
Bcl11a siRNA-2292 restraining expression of BCL11A and proliferation of tumorous B cells and application thereof Download PDFInfo
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Abstract
The invention discloses a Bcl11a siRNA-2292 restraining the expression of BCL11A and the proliferation of tumorous B cells, which has the sense strand sequence of 5'-CGGGCGAAAGG CCUUAUAAUU-3' and the antisense strand sequence of 5'-UUAUAAGGCCUUU CGCCCGUG-3'. The Bcl11a siRNA-2292 can efficiently restrain the expression of the BCL11A, effectively restrain the proliferation of the tumorous B cells and promote the apoptosis of the tumorous B cells. The invention also confirms that the combination of the Bcl11a siRNA-2292 with the Bcl-2siRNA can more effectively restrain the proliferation and promote the apoptosis of the tumorous B cells. The Bcl11a siRNA-2292 restraining the expression of the BCL11A and the proliferation of the tumorous B cells has important meanings in developing novel anti-B cell oncogene medicines and improving the treatment effect of B cell tumors.
Description
Technical field
The present invention relates to siRNA and Application Areas thereof, be specifically related to the Bcl11a siRNA-2292 of a kind of BCL11A of inhibition expression and tumprigenicity B cell proliferation and treat and/or prevent the application in the B cell tumour medicine in preparation.
Background technology
The RNAi technology is a kind of new way of the inhibition of gene expression of high specific efficiently.The research of inside and outside has shown that the RNAi technology has the better application prospect than antisense nucleic acid aspect gene therapy for cancer.A large amount of researchs show that external expression through RNAi inhibition tumor-related gene can suppress propagation, the growth of tumour cell, and cell death inducing increases the susceptibility of tumour cell to medicine.
Bcl11a (the B-cell lymphoma/leukemia 11A) assignment of genes gene mapping is that a kr ü ppel appearance zinc refers to gene in 2p16.1, belongs to BCL11 family (comprising BCL11A and BCL11B).The Bcl11a gene has 5 exons, mainly contains 4 kinds of transcripts and can produce corresponding 4 kinds of major protein (XL, 5.9kb/125kD; L, 3.8kb/100kD; S, 2.4kb/35KD; XS; 1.5kb/25KD); Exons 1 and 2 is that 4 kinds of phenotypes are common, and promptly all phenotypes of BCL11A have common N-terminal [Liu H, Ippolito GC; Wall JK, et al.Functional studies of BCL11A:characterization of the conserved BCL11A-XLsplice variant and its interaction with BCL6 in nuclear paraspeckles of germinal center B cells.Mol Cancer.2006; 5:18].No matter in normal Lymphoid tissue still in lymphoma, BCL11A-XL is main expressing protein.Research at present shows; BCL11A presents high expression level [Weniger MA at the multiple B cell tumour of the mankind; Pulford K; Gesk S, et al.Gains of the proto-oncogene BCL11A and nuclear accumulation of BCL11A (XL) protein are frequent in primary mediastinal B-cell lymphoma.Leukemia.2006; 20 (10): 1880-1882]; In the normal lymphoglandula of human body, marrow, spleen, faint expression is only arranged; And mainly be expressed in the B of germinal center cell, and in most of human body healthy tissues, do not express, prompting BCL11A maybe be relevant with the biological characteristics of B cell tumour.In fact; It is unusual to have shown that BCL11A can occur through gene amplification, chromosome translocation or gene activation in the multiple B cell tumour of the mankind; Effect [the Yin B that serves as a proto-oncogene; Delwel R, Valk PJ, et al.A retroviral mutagenesis screen reveals strong cooperation between Bcl11a overexpression and loss of the Nf1 tumor suppressor gene.Blood.2009; 113 (5): 1075-1085].
Existing research shows that BCL11A maybe be relevant with the generation of B cell tumour, but does not also have Bcl11a siRNA to can be used for tumour at present, especially the evidence that treats and/or prevents of B cell tumour.
Summary of the invention
The shortcoming that primary and foremost purpose of the present invention is to overcome prior art provides the Bcl11a siRNA-2292 of a kind of BCL11A of inhibition expression and tumprigenicity B cell proliferation with not enough.
Another object of the present invention is to provide described inhibition BCL11A to express and the application of the Bcl11a siRNA-2292 of tumprigenicity B cell proliferation.
The object of the invention is realized through following technical proposals: a kind of Bcl11a siRNA-2292 that suppresses BCL11A expression and tumprigenicity B cell proliferation, and sequence is as follows:
The sequence of positive-sense strand is: 5 '-CGGGCGAAAGGCCUUAUAAUU-3 ';
The sequence of antisense strand is: 5 '-UUAUAAGGCCUUUCGCCCGUG-3 ';
Said inhibition BCL11A expresses and the Bcl11a siRNA-2292 of tumprigenicity B cell proliferation treats and/or prevents the application in the B cell tumour medicine in preparation;
Described B cell tumour comprises B cell leukemia and B cell lymphoma.
A kind of B cell tumour medicine that treats and/or prevents comprises outside the Bcl11a siRNA-2292 of said inhibition BCL11A expression and tumprigenicity B cell proliferation, also can comprise Bcl-2siRNA;
The sequence of described Bcl-2 siRNA is as follows:
The sequence of positive-sense strand is: 5 '-GUACAUCCAUUAUAAGCUGUU-3 ';
The sequence of antisense strand is: 5 '-CAGCUUAUAAUGGAUGUACUU-3 ';
The present invention has following advantage and effect with respect to prior art:
(1) Bcl11asiRNA-2292 of inhibition BCL11A of the present invention expression and tumprigenicity B cell proliferation can efficiently suppress the BCL11A expression.
(2) inhibition of the present invention BCL11A express and the Bcl11asiRNA-2292 of tumprigenicity B cell proliferation effectively inhibition of enoplastic B cell propagation and promote its apoptosis.
(3) Bcl11asiRNA-2292 of inhibition BCL11A of the present invention expression and tumprigenicity B cell proliferation is significant with the result of treatment that improves the B cell tumour for the new anti-B cell oncogene medicine of exploitation.Environment for human survival goes from bad to worse, and B cell tumour (comprising B cell leukemia and B cell lymphoma) M & M is increasingly high, has become one of main neoplastic disease that influences health of people and life-span.Most B cell tumours all has BCL11A to express to be increased, thereby the treatment of target BCL11A will have commonplace meaning, is applicable to most B cell tumours, will have huge market potential.
(4) the present invention confirms that described inhibition BCL11A expresses the Bcl11asiRNA-2292 and Bcl-2 siRNA combination with the tumprigenicity B cell proliferation, the propagation of more effective inhibition of enoplastic B cell and promote its apoptosis.
Description of drawings
Fig. 1 is that flow cytometer detects cell transfecting efficient (* 100) behind the transfectional cell 6h, wherein:
A is the SUDHL6 cell of untransfected; B is the SUDHL6 cell of the fluorescently-labeled negative siRNA of transfection; C is the EB1 cell of untransfected; D is the EB1 cell of the fluorescently-labeled negative siRNA of transfection.
Fig. 2 is that the Bcl11a mRNA in the SUDHL6 cell expresses spirogram behind the transfection Bcl11a siRNA-2292 48h.
Fig. 3 is that the Bcl11a mRNA in the EB1 cell expresses spirogram behind the transfection Bcl11a siRNA-2292 48h.
Fig. 4 is the influence figure of Bcl11a siRNA-2292 to SUDHL6 cell BCL11A protein expression, and wherein: swimming lane 1 is irrelevant sequence set; Swimming lane 2 is the idle running group; Swimming lane 3 is a groups of cells; Swimming lane 4 is the Bcl11asiRNA-2292 group.
Fig. 5 is the influence figure of Bcl11a siRNA-2292 to EB1 cell BCL11a protein expression, and wherein: swimming lane 1 is Bcl11a siRNA-2292 group; Swimming lane 2 is irrelevant sequence set; Swimming lane 3 is the idle running group; Swimming lane 4 is a groups of cells.
Fig. 6 is the apoptotic morphology figure of SUDHL (Hoechst, * 200).
Fig. 7 is the apoptotic morphology figure of EB1 (Hoechst, * 200).
Fig. 8 is SUDHL apoptosis rate figure behind the transfection Bcl11a siRNA-2292 72h.
Fig. 9 is EB1 apoptosis rate figure behind the transfection Bcl11a siRNA-2292 72h.
Figure 10 is SUDHL6 apoptosis figure behind the flow cytometer AnnexinV/PI method detection transfection Bcl11a siRNA-2292 72h, and wherein: A is a groups of cells; B is the idle running group; C is irrelevant sequence set; D is the siRNA-2292 group.
Figure 11 is EB1 apoptosis figure behind the flow cytometer AnnexinV/PI method detection transfection Bcl11a siRNA-2292 72h, and wherein: A is a groups of cells; B is the idle running group; C is irrelevant sequence set; D is the siRNA-2292 group.
Embodiment
Below in conjunction with embodiment and accompanying drawing the present invention is described in further detail, but embodiment of the present invention is not limited thereto.
One, experiment material and method
(1) design of Bcl11a siRNA and synthetic
The design tool software (referring to www.ambion.com/techlib/misc/siRNA finder.html) that provides on the net according to Ambion company is directed against 3 siRNA sequences of Bcl11a gene design; Difference called after Bcl11a siRNA-2292, Bcl11a siRNA-475 and Bcl11a siRNA-319; Design the negative siRNA of positive-sense strand 5 ' end green fluorescence FAM mark and the sequence siRNA that has nothing to do simultaneously as contrast, synthetic by Shanghai JiKai Gene Chemical Technology Co., Ltd.
The sequence of Bcl11a siRNA-2292 is following:
The sequence of positive-sense strand is: 5 '-CGGGCGAAAGGCCUUAUAAUU-3 ';
The sequence of antisense strand is: 5 '-UUAUAAGGCCUUUCGCCCGUG-3 ';
The sequence of Bcl11a siRNA-475 is following:
Positive-sense strand: 5 '-GCAGGGUAUUUGUAAAGAUUU-3 ';
Antisense strand: 5 '-AUCUUUACAAAUACCCUGCGG-3 ';
The sequence of Bcl11a siRNA-319 is following:
Positive-sense strand: 5 '-AGAAGAUGACGAUUGUUUAUU-3 ';
Antisense strand: 5 '-UAAACAAUCGUCAUCUUCUGG-3 ';
The sequence of fluorescently-labeled negative siRNA is following:
Positive-sense strand: 5 '-UUCUCCGAACGUGUCACGUUU-3 ';
Antisense strand: 5 '-ACGUGACACGUUCGGAGAAUU-3 ';
The sequence of irrelevant sequence siRNA is following:
Positive-sense strand: 5 '-GUAUGACAACAGCCUCAAGUU-3 ';
Antisense strand: 5 '-CUUGAGGCUGUUGUCAUACUU-3 '.
(2) cell cultures
Diffuse large B lymphoma cell line SUDHL6 (U.S. ATCC cell bank) and Burkitt lymphoma (Burkitt lymphoma) clone EB1 (U.S. ATCC cell bank) are inoculated in respectively in the RPMI1640 substratum that contains volume(tric)fraction 10% NBCS, 100U/mL penicillium mould and 100U/mL Streptomycin sulphate, are containing volume(tric)fraction 5%CO
237 ℃ of cultured continuously of incubator.
(3) cell transfecting
1. transfection previous day, cell density is transferred to 2 * 10
5/ ml is resuspended in 24 orifice plates then, and every hole adds the cell suspension of 0.5ml, 37 ℃, 5%CO
2Cultivate in the incubator.
2. transfection same day, the cell in every hole in 24 orifice plates is resuspended in the RPMI1640 substratum that 100 μ l contain volume(tric)fraction 10% NBCS.
3. do not contain the RPMI1640 substratum dilution siRNA of serum with 100 μ l, the final concentration of siRNA is 100nM.Add 6 μ l Hiperfect transfection reagents again, mixing.Incubated at room mixed solution 5～10 minutes.Above mixed solution is the amount in the every hole of 24 orifice plates.
4. ready mixed solution joins respectively in the cell suspension more than inciting somebody to action, wave and culture plate, light mixing.37 ℃, 5%CO
2Cultivate in the incubator.
5. behind the 6h, every hole adds the RPMI1640 substratum that 400 μ l contain volume(tric)fraction 10% NBCS, 37 ℃, 5%CO
2Cultivate in the incubator.
The detection of transfection efficiency: transfection 6h, the transfection situation of observation of cell under fluorescent microscope, and through flow cytometer detection by quantitative cell transfecting efficient (transfection efficiency is to detect with the fluorescently-labeled negative siRNA of transfection).
(4) fluorescence quantitative RT-RCR detects transfectional cell Bcl11a mRNA expression level
Blank control group, the simple i.e. idle running group of transfection reagent group, irrelevant sequence set, Bcl11a siRNA-475 group, Bcl11a siRNA-319 group and Bcl11a siRNA-2292 are organized cell with (4～8) * 10
5/ mL initial concentration is inoculated in 6 orifice plates, and every hole inoculation 1mL carries out the transfection continued by the explanation of HiPerfect transfection reagent box and cultivates 48h, centrifugal collecting cell.RNA extract to use the RNAzol test kit (Gibco, BRL) and use random primer (USA) cDNA first chain is synthesized in reverse transcription for Superscript II Kit, Invitrogen with the ThermoScript II test kit.And through the definite quality of synthesizing cDNA of GAPDH Gene RT-PCR.All undertaken by ordinary method, concrete extractive process is described below.
1) extraction of cell total rna and purifying
1. collect eccentric visual cell, remove its supernatant, with PBS (0.01M:NaCl 8g, KCl 0.2g, Na
2HPO
41.44g, KH
2PO
40.24g, pH7.4) wash twice after, cell transfer is gone in the 1.5mL centrifuge tube, every pipe adds Trizol reagent 1mL, shakes up, leave standstill 15min under the room temperature after, blow and beat lysing cell repeatedly;
2. the good cell pyrolysis liquid of digestion in each pipe is drawn onto during 1.5mL EP that DEPC handled manages, adds chloroform 0.2mL (Trizol: chloroform is about 5: 1), jog 15s, and hatch 15min on ice;
3. 4 ℃, 12, the centrifugal 15min of 000rpm.Get the colourless water of supernatant then to the EP pipe that DEPC handled, add the 0.5mL Virahol, leave standstill 10min under the room temperature;
4. 4 ℃, 12, the centrifugal 10min of 000rpm.Observe total RNA in the pipe white precipitate at the end, supernatant discarded;
5. add the ethanolic soln 1.0mL that newly prepares with DEPC water, washing precipitation gently, 4 ℃, 12, the centrifugal 5min of 000rpm through the volume percent 75% of precooling;
6. remove supernatant, instantaneous centrifugal, blot liquid with little Tip; Make the deposition seasoning, DEPC treating water 20～30 μ L add, mixing, and 55～60 ℃ of water-bath 10min dissolve total RNA;
7. survey total RNA purity and concentration with the uv-spectrophotometric appearance, identify whether hydrolysis of RNA, identify that the back is subsequent use in-70 ℃ of preservations with agarose gel electrophoresis.
2) evaluation of cell total rna
Concentration analysis and purity are identified: from the RNA that extracts, draw 1 μ L, be diluted to 100 μ L with DEPC water, survey the concentration of total RNA, the ratio of absorbance A260 and A280 (A260/A280) respectively with the uv-spectrophotometric appearance.
3) reverse transcription reaction
Get above-mentioned RNA sample, it be diluted to 1 μ g/ μ L, in the PCR pipe, add the mixture of following reagent:
On the PCR appearance according to following conditioned response: 30 ℃ of 10min, 42 ℃ of 20min, 99 ℃ of 5min, 4 ℃ of 5min, instantaneous centrifugal ,-20 ℃ of preservations are subsequent use.
4) pcr amplification
The real-time quantitative PCR test kit is purchased the root biotech firm in sky, Beijing.Real-time quantitative PCR reaction tubes and reaction kit are U.S. BIO-RAD Company products.The Bcl11a primer sequence is following: upstream primer: 5 '-AACCCCAGCACTTAAGCAAA-3 '; Downstream primer: 5 '-GGAGGTCATGATCCCCTTCT-3 '.With GAPDH is internal reference, and upstream primer is 5 '-ACCCAGAAGACTGTGGATGG-3 ', and downstream primer is 5 '-TTCAGCTCAGGGATGACCTT-3 '.
Utilize SYBR Green I dyestuff to detect cell Bcl11a expression, and with GAPDH as internal reference.Total reaction volume is 20 μ L.Reaction conditions: after 95 ℃ of 10min sex change, carry out 40 cyclic amplifications altogether, each circulation comprises 95 ℃ of 15s, 60 ℃ of 30s and 80 ℃ of 5s, and read plate 1 time at 80 ℃.Subsequently,, every with 0.17 ℃/s pace of change at a distance from 1 fluorescent value of 2s record from 65 ℃ to 95 ℃, obtain melting curve.Institute's amplification PCR products melting curve is analyzed, carried out mass volume ratio 2% agarose gel electrophoresis simultaneously at random, to confirm whether product is the purpose fragment that is increased.Adopt the relative quantification formula: 2-Δ Ct * 100%, Δ Ct=Ct (Bcl11a)-Ct (GAPDH), the relative quantity of calculating cell Bcl11a.
5) Western blot detects the proteic expression of BCL11A
Collection is through the cell of transfection 48h, and the centrifugal collecting cell sample adopts tenuigenin and nucleoprotein to extract test kit in the 1.5ml centrifuge tube, extracts total protein of cell, and the BCA method is carried out quantitatively.Get 100 μ g albumen and sample loading buffer (1.0M pH6.8 Tris-Hcl 1.0ml, 10%SDS 6.0ml, beta-mercaptoethanol 0.2ml, distilled water 2.8ml) and mix 100 ℃ of sex change 5min; Mass percent 12%SDS-PAGE gel electrophoresis separates; The fine film of electrotransfer to nitre, GAPDH one is anti-as internal reference, and film is 4 ℃ of incubated overnight in one resists; The two anti-2h, ECL color developing detection of hatching.Detailed process is following:
1. lysing cell extracts total protein
A, get 10
6Individual cell adds 1mL protein lysate RIPA and 10mL PMSF (PMSF), ice bath 30min, 4 ℃, the centrifugal 30min of 13000g.
B, supernatant is carefully transferred in the clean aseptic centrifuge tube-20 ℃ of preservations.
2. electrophoresis and hybridization
A, preparation mass percent 12%Tris-glycocoll sds polyacrylamide gel electrophoresis separation gel solution; The perfusion acrylamide soln; The complete hypsokinesis of separation gel polymerization goes out to cover water layer; Mass percent 5% is concentrated after glue pours into, get clean comb and be inserted into and concentrate in the glue, gel is placed room temperature.
B, get an amount of sample and join in equal-volume 2 * sds gel sample loading buffer, boil 5min in the boiling water and make protein denaturation, go up appearance behind the mixing.
C, with careful the shifting out of comb, gel sets at electrophoresis apparatus, last appearance.Energized, voltage are 60V.
After D, electrophoresis finish, separation gel is downcut, make the structure of filter paper-gel-film-filter paper, change film.Voltage 300mA during transfer, transfer time 1h.
E, shift to finish after, put into the skim-milk confining liquid that fills mass percent 5% to pvdf membrane, sealing 2h or spend the night.
F, sealing finish the back with contain the pH7.4 of 1g/L Tween20, the Tris hydrochloride buffer (TBST) of 50mmol/L is washed film 5 times, each 5 minutes, adding one, anti-(BCL11A press mass volume ratio mg/ml dilutes at 1: 500; GAPDH presses mass volume ratio mg/ml dilution at 1: 1000).4 ℃ of incubated overnight are washed film 5 times, each 5min.
G, add horseradish peroxidase-labeled two anti-(press at 1: 1000 mass volume ratio mg/ml dilution), reaction 1h washes film 5 times, at every turn 5min.
H, pvdf membrane is soaked in the ECL fluorogenic substrate, behind incubated at room 3～5min, be placed on the darkroom, get exograph, behind the exposure 1min, development, photographic fixing successively, take pictures.
6) the CCK8 method is surveyed inhibitory rate of cell growth
The SUDHL6 cell and the EB1 cell of logarithmic phase are inoculated in respectively in 96 well culture plates, carry out transfection, place 37 ℃, saturated humidity, 5%CO according to above-mentioned steps (step (3))
2Behind conventional cultivation 24,48 and the 72h, in every hole, add 30 μ L CCK8 under the condition, place 37 ℃ to hatch 4h again.Directly join 450nm wavelength survey A450 value on the detector then, reflect cell survival quantity indirectly with A450 at enzyme.Experiment repetition 3 times can be calculated the inhibiting rate of each time point pair cell after the Bcl11a siRNA transfection in view of the above.
7) Hoechst dyeing observing apoptosis cellular form
Collection is through the cell of transfection 72h, and the centrifugal collecting cell sample adds the 0.5ml stationary liquid in the 1.5ml centrifuge tube, mixing, fixing 10 minutes.The centrifugal stationary liquid that goes, the PBS of 0.01M, pH 7.2 washes twice.Last centrifugal back is inhaled and is gone most of liquid to keep about 50 μ l liquid, has slowly hanged cell again, drops on the slide glass, dries, and evenly drips and goes up 0.5ml Hoechst 33258 staining fluids, dyes 5 minutes, dries, and PBS washes twice.Drip anti-fluorescent quenching mounting liquid on slide glass, cover a clean deckglass, avoid bubble.Fluorescent microscope can detect the nucleus that is blue.
8) two method and the flow cytometer detection apoptosis rates of dying of AnnexinV/PI
48h's respectively organizes cell, the PBS washing of 0.01M, pH 7.2 2 times, the centrifugal supernatant that goes after the centrifugal collection transfection; 195 μ L Annexin V-FITC combine the liquid re-suspended cell, add Annexin V-FITC 5 μ L, and lucifuge is hatched 10min; The centrifugal supernatant that goes, 190 μ L Annexin V-FITC combine the liquid re-suspended cell, add 10 μ L propidium iodide (PI) staining fluids; The ice bath lucifuge is placed, and carries out flow cytometer immediately and detects, with MULTCYCLE software analysis result.
9) statistical procedures
Adopt SPSS13.0 software to carry out the statistical analysis of data.Experimental data representes that with means standard deviation data relatively adopt the one-way analysis of variance of block design fully immediately between many groups, and relatively selecting for use between group can be carried out mean S-N-K check relatively in twos between a plurality of sample averages.
Two, experimental result
(1) Bcl11a siRNA suppresses SUDHL6 and EB1 growth of tumour cell
(1) cell transfecting efficient
The cell of the fluorescently-labeled negative siRNA 6h of transfection is placed observation under the inverted fluorescence microscope; Can see the parts of fine born of the same parents and present the green fluorescence phenomenon; It is 46.2% that flow cytometer detects SUDHL6 cell transfecting efficient, and EB1 cell transfecting efficient is 51.4% (as shown in Figure 1).The experiment of back is all carried out transfection by this transfection conditions.
(2) fluorescence quantitative RT-RCR detects cell Bcl11a mRNA expression level
The total RNA that extracts 1.8～2.0, explains that the RNA purity of extracting is higher through the ratio of its absorbance A of UV spectrophotometer measuring 260/A280.
At first; In 3 Bcl11a siRNA, filter out the expression of siRNA-2292 transfection SUDHL6 cell 48h downward modulation Bcl11a mRNA; Its value significantly is lower than blank control group (promptly referring to the groups of cells without any processing), idle running group (promptly referring to simple transfection reagent treatment group) and irrelevant sequence set (P＜0.05), and other 2 siRNA (Bcl11a siRNA-475 and Bcl11a siRNA-319), idle running group and irrelevant sequence siRNA organize respectively and do not have significant difference (P＞0.05) between the Bcl11a mRNA expression amount of blank control group cell.Equally, the expression of siRNA-2292 transfection EB1 cell 48h downward modulation Bcl11a mRNA, visible, but Bcl11a siRNA-2292 specificity suppresses Bcl11a expression of gene (result is shown in Fig. 2 and 3) in SUDHL6 cell and the EB1 cell.
(3) detection of BCL11A protein expression
The BCL11A expressing quantity of SUDHL6 cell and EB1 cell 48h cell behind transfection Bcl11a siRNA-2292 reduces; And significantly be lower than control group (i.e. idle running group, irrelevant sequence set and blank control group), and idle running group and irrelevant sequence set are respectively and do not see significant difference between blank control group.Show, but Bcl11a siRNA-2292 specificity suppresses the proteic expression of BCL11A (result is shown in Figure 4 and 5) in SUDHL6 and the EB1 cell.
(4) Bcl11a siRNA-2292 is to the influence of SUDHL6 and the growth of EB1 cell
CCK8 result shows: the proliferation activity of Bcl11a siRNA-2292 transfection group cell is lower; And significantly be lower than other control group (i.e. idle running group, irrelevant sequence set and blank control group) (P＜0.05), and idle running group and irrelevant sequence set do not have significance (P＞0.05) (seeing table 1 and 2) with the multiplication capacity differences of blank control group cell respectively.It is thus clear that Bcl11a siRNA-2292 can suppress the growth of SUDHL6 and EB1 cell.
Each time point CCK8 method detects SUDHL6 cell absorbance A value (
n=6) behind the table 1 transfection BCL11a siRNA
Compare * P＜0.05 with other groups
Each time point CCK8 method detects EB1 cell absorbance A value (
n=6) behind the table 2 transfection Bcl11a siRNA
Compare * P＜0.05 with other groups
(5) Hoechst dyeing detects the apoptosis form
Fig. 6 and 7 Hoechst colored graphs for 72h after the transfection.The all visible obvious apoptosis cell of Bcl11a siRNA-2292 group: intact nuclear membrane, smaller volume, caryoplasm pyknosis; And other groups (i.e. idle running group, irrelevant sequence set and blank control group) are not seen obvious apoptotic cell.
(6) flow cytometer detects apoptosis rate
Flow cytometer-AnnexinV/PI is two to be dyed Faxian and shows that transfection Bcl11a siRNA-2292 goes into that total apoptosis rate of 72h increases behind the SUDHL6 cell, has significance (P＜0.05) with blank group and irrelevant sequence set comparing difference.And the apoptosis rate difference of blank group and irrelevant sequence set does not have significance (P＞0.05), sees Fig. 8.Transfection Bcl11a siRNA-2292 goes into that the apoptosis rate of 72h is significantly higher than blank group and irrelevant sequence set (P＜0.05) behind the EB1 cell, and blank group nothing to do with sequence set differences does not have significance (P＞0.05), sees Fig. 9.It is thus clear that Bcl11a siRNA-2292 can induce the apoptosis rate (Figure 10,11) that improves SUDHL6 and EB1 cell.
Three, Bcl11a siRNA-2292 and Bcl-2siRNA unite the restraining effect of enhancing to the B growth of tumour cell
(1) Bcl11a siRNA-2292 and Bcl-2siRNA unite the influence to the B growth of tumour cell
The CCK8 method shows; Transfection Bcl11a siRNA-2292 and Bcl-2siRNA (1: 1 in molar ratio ratio collocation between Bcl11a siRNA-2292 and the Bcl-2siRNA simultaneously; Final concentration is 50nM) go into SUDHL6 and EB1 cell after; Each time point Bcl11a siRNA-2292 and Bcl-2siRNA group are single organizes the growth that (final concentration is 100nM) obviously suppressed cell with Bcl11a siRNA-2292 group (final concentration is 100nM), list with Bcl-2siRNA, and the result shows that Bcl11a siRNA-2292 and Bcl-2siRNA associating can strengthen the inhibition (seeing table 3 and 4) of cell growth.
The CCK8 method detects SUDHL6 cell absorbance A value
n=6 behind the table 3 Bcl11a siRNA associating Bcl-2siRNA)
* compare P＜0.05 with other groups
The CCK8 method detects EB1 cell absorbance A value
n=6 behind the table 4 Bcl11a siRNA associating Bcl-2siRNA)
* compare P＜0.05 with other groups.
(2) Bcl11a siRNA-2292 and Bcl-2siRNA unite the influence to the B apoptosis of tumor cells
Flow cytometer-AnnexinV/PI pair of stainings detection is as shown in table 5, and the inductive apoptosis rate is all organized with Bcl-2siRNA with Bcl11a siRNA-2292 group, list apparently higher than single behind Bcl11a siRNA-2292 and Bcl-2siRNA group combined action SUDHL6 and the EB1 cell.
Table 5 transfection Bcl11a siRNA associating BCL2-siRNA72h apoptosis rate
* compare P＜0.05 with other groups
The foregoing description is a preferred implementation of the present invention; But embodiment of the present invention is not restricted to the described embodiments; Other any do not deviate from change, the modification done under spirit of the present invention and the principle, substitutes, combination, simplify; All should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (5)
1. one kind is suppressed that BCL11A expresses and the Bcl11a siRNA-2292 of tumprigenicity B cell proliferation, it is characterized in that sequence is as follows:
The sequence of positive-sense strand is: 5 '-CGGGCGAAAGGCCUUAUAAUU-3 ';
The sequence of antisense strand is: 5 '-UUAUAAGGCCUUUCGCCCGUG-3 '.
2. the application of the Bcl11a siRNA-2292 of the said inhibition of claim 1 BCL11A expression and tumprigenicity B cell proliferation is characterized in that: described Bcl11a siRNA-2292 is used for preparation treatment B cell tumour medicine.
3. according to the said application that suppresses the Bcl11a siRNA-2292 of BCL11A expression and tumprigenicity B cell proliferation of claim 2, it is characterized in that: described B cell tumour is B cell leukemia and/or B cell lymphoma.
4. treat B cell tumour medicine for one kind, it is characterized in that comprising the Bcl11a siRNA-2292 of the described inhibition of claim 1 BCL11A expression and tumprigenicity B cell proliferation.
5. treatment B cell tumour medicine according to claim 4 is characterized in that: also comprise Bcl-2 siRNA;
The sequence of described Bcl-2 siRNA is as follows:
The sequence of positive-sense strand is: 5 '-GUACAUCCAUUAUAAGCUGUU-3 ';
The sequence of antisense strand is: 5 '-CAGCUUAUAAUGGAUGUACUU-3 '.
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