CN103614379A - FoxQ1 gene-targeting interfering siRNA (Small Interfering Ribonucleic Acid) and application thereof to resisting nonsmall-cell lung cancer - Google Patents

Abstract

The invention discloses a FoxQ1 gene-targeting interfering siRNA (Small Interfering Ribonucleic Acid) and an application thereof. The siRNA has the sequence as follows: a positive-sense strand: 5'-GCCAAGCAAUUUCUUUAAATT-3', and an antisense strand: 5'-UUUAAAGAAAUUGCU UGGCTT-3'. The FoxQ1 gene-targeting interfering siRNA is successfully constructed by using modern molecular biological techniques such as fluorescent quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction), Western Blot, a CCK8 method, a Transwell cell model, flow cytometry and the like; the possibility for treating lung cancer by taking FoxQ1 as a target point and the effect of treating tumor through assisting chemotherapy are determined through researching the influence to proliferation, invasion, migration, apoptosis and chemotherapy sensitivity of the lung cancer cells and an inhibiting effect for the tumor forming ability of a naked mouse on cell and animal levels after an FoxQ1 gene is interfered; the siRNA has a wide application prospect in specifically silencing the FoxQ1 gene.

Description

A kind of interference siRNA of target FoxQ1 gene and the purposes of anti-nonsmall-cell lung cancer thereof

Technical field

The invention belongs to biological medicine technology field, be specifically related to a kind of interference siRNA of target FoxQ1 gene and the purposes of anti-nonsmall-cell lung cancer thereof.

Background technology

Lung cancer is one of incidence and the highest malignant tumour of mortality ratio in the world today, approximately there are in the world every year 1350000 people to be diagnosed as lung cancer, in recent years, the death toll that lung cancer causes has surpassed mammary cancer, colorectal cancer, carcinoma of the pancreas etc., becomes the first cause of cancer mortality.Wherein nonsmall-cell lung cancer (NSCLC) is modal Lung Cancer Types, and considerable patient belongs to the intractable lung cancer invalid to traditional treatment.Although the progress of the pattern of multidisciplinary synthesis treatment is in recent years enhanced the result for the treatment of of nonsmall-cell lung cancer, due to reasons such as transfer and resistances, mortality ratio is still very high at present, and total five year survival rate is 15% left and right only.Therefore in the urgent need to seeking new diagnosis index and the treatment of strengthening comprehension, explore new therapeutic strategy and treatment target spot, become lung cancer and study one of field of greatest concern.

The generation development of tumour is the pathologic process of a multistage, multi-step, polygene participation, relates to the unconventionality expression of the activation of proto-oncogene, the inactivation of cancer suppressor gene and apoptosis-related genes, is the abnormal disease of a kind of polygene in itself.RNA disturbs (RNA interference, RNAi) be development and gradually an emerging gene silent technology of maturation increasingly at present, endogenous or exogenous double-stranded RNA (double strand ed RNA, dsRNA) on the post-transcriptional level of gene, in mediated cell there is specificity degraded in mRNA, cause expression of target gene reticent, thereby produce the disappearance of corresponding function phenotype, that specificity suppresses to have the process with the expression of the gene of dsRNA homologous sequence, belong to sequence-specific PTGS, there is high efficiency, specificity, position effect, the feature of competitive effect and propagability.Quantity research shows greatly, and RNAi is the ancient phenomenon that is extensively present in organic sphere, and different organisms comprises in plant, fruit bat, nematode and some mammalian cells and all has RNAi phenomenon.RNAi guards in organic evolution; it is the protective mechanism that organism is resisted virus or the invasion of other external nucleic acid and keeps self inheritance stability; be comprise the mankind from lower eukaryotes to mammalian body in naturally occurring gene silencing mechanism; its efficiency that suppresses expression of target gene is high more than antisense nucleic acid, and the time length is also longer.Do not affecting under the prerequisite of normal gene function, RNA disturbs can suppress sudden change or the overexpression of the important gene in some Carcinogenesis, thereby reaches gene therapy purpose.Constantly perfect along with RNA perturbation technique, has obtained sure effect for the RNAi of various malignant tumours.The gene therapy research that is found to be of RNAi phenomenon provides strong instrument.SiRNA, compares with antisense nucleic acid apparently higher than antisense nucleic acid the suppression efficiency of target gene, and siRNA consumption is lower, and has overcome some shortcomings of antisense nucleic acid, as the non-specific binding of oligomer, easy degraded etc.The siRN of chemosynthesis can proceed to fast in cell and have an effect, and meanwhile, the siRNA expense of chemosynthesis is high, easily degraded, and transient expression only, and its left and right can only continue week age.Be unfavorable for further steady in a long-termly carrying out mechanism research.

Forkhead (Fox) transcription factor family member transcribes by recruiting co-activation factor regulatory gene, participate in the regulate several biological processes such as Growth of Cells, differentiation, apoptosis, migration, its sudden change occurs relevant with abnormal expression with developmental malformation, metabolic disease and tumour.FoxQ1 is one of member that Fox transcription factor family is newer, is positioned at human chromosomal 6p23-25, and early stage research finds that FoxQ1 is relevant with hair follicle development and cytodifferentiation; Now be proved in some malignant tumour of the mankind develops and played the part of very important role.In addition, FoxQ1 is raised by transforming growth factor-beta (transforming growth factor-β 1, TGF-β 1) induction, participates in epithelial cell form and reinvents and break up.Current research finds that FoxQ1 expresses and significantly increases in intestinal cancer tissue, and the researchs such as Kaneda show the expression of FoxQ1 regulation and control cancer suppressor gene p21, in occurring, develop, plays an important role in intestinal cancer.Zhang etc. report most high expression level FoxQ1 of breast cancer tissue, cross in vitro expression FoxQ1 in breast cancer cell, can promote cell proliferation and clone to form, and strengthen cancer cells invasiveness, cross in vivo expression FoxQ1 and can promote breast cancer cell lung to shift.

In the research Zhong， of lung cancer seminar, in previous experiments, confirmed and reported (P MID:22761930 in PLOS ONE magazine; Doi:10.1371), FoxQ1 is high expression level in most of nonsmall-cell lung cancers (NSCLC) tissue, and closely related with malignancy and patient's prognosis, patient's five year survival rate of FoxQ1 high expression level is starkly lower than the patient of low this gene of expression.Therefore, use RNA to disturb Fo xQ1 in NSCLC is suppressed, can suppress the growth of NSCLC, the biological characteristics of NSCLC is exerted an influence.

Summary of the invention

Goal of the invention: for the deficiencies in the prior art, the object of this invention is to provide a kind of target Fox Q1 gene and disturb small molecules siRNA, specifically reticent FoxQ1 gene.Another object of the object of the invention is to provide the carrier that contains this interference small molecules siRNA.The present invention also has an object to be to provide the application of this interference small molecules siRNA.

The present invention is directed to the siRNA sequence of FoxQ1, can be chemosynthesis can be also by expression vector stably express.For the siRNA sequence of FoxQ1, by forms such as liposome, bioabsorbable carrier material, virus vector, carry siRNA sequence.The present invention is directed to the coding region sequence of FoxQ1 gene, according to corresponding siRNA principle of design, design a pair of siRNA sequence, and by transfected with human lung adenocarcinoma cell, detected the silence efficiency to FoxQ1 gene, confirm its validity and specificity.Then effective siRNA fragment is designed to insert the hair clip siRNA Insert Fragment of pGPU6/GFP/Neo carrier, the expression vector that structure can stably express shRNA.

Technical scheme: for achieving the above object, the technical solution used in the present invention is as follows:

Target FoxQ1 gene disturbs a siRNA, and the sequence of described siRNA is as follows:

Positive-sense strand: 5 '-GCCAAGCAAUUUCUUUAAATT-3 ',

Antisense strand: 5 '-UUUAAAGAAAUUGCUUGGCTT-3 '.

A kind of clone has described target FoxQ1 gene to disturb the carrier of siRNA.

The construction process of described carrier: the siRNA sequence of reticent FoxQ1 gene is specifically designed to be able to the hair clip siRNA Insert Fragment in insertion vector, then be connected with pGPU6/GFP/Neo carrier, obtain the interference microRNA for anti-nonsmall-cell lung cancer biological behaviour.

Described target FoxQ1 gene disturbs the application of siRNA in reticent FoxQ1 gene specifically.

Beneficial effect: compared with prior art, the present invention uses fluorescence quantitative RT-RCR, Western Blot, CCK8 method, Transwell cell model, the modern molecular biology techniques such as flow cytometry, successfully construct target FoxQ1 gene and disturb siRNA, by after disturbing FoxQ1 gene in cell levels and the research of animal level to proliferation of lung cancer cells, invasion and attack, migration, the impact of apoptosis and chemosensitivity, and nude mice is become to the restraining effect of knurl ability, determine and to using FoxQ1 as the possibility of target treatment lung cancer and to assist the effect of chemotherapeutic treatment tumour, this siRNA being with a wide range of applications in reticent FoxQ1 gene specifically.

Accompanying drawing explanation

Fig. 1 is the FoxQ1 gene expression results figure of the expression level of FoxQ1 gene mRNA and albumen in four strain lung adenocarcinoma cells;

Fig. 2 is the solubility curve figure of the expression level of FoxQ1 gene mRNA and albumen in four strain lung adenocarcinoma cells;

Fig. 3 is the amplification curve diagram of the expression level of FoxQ1 gene mRNA and albumen in four strain lung adenocarcinoma cells;

Fig. 4 is the FoxQ1 protein expression figure of the expression level of FoxQ1 gene mRNA and albumen in four strain lung adenocarcinoma cells;

Fig. 5 is the gray-scale value scintigram of the expression level of FoxQ1 gene mRNA and albumen in four strain lung adenocarcinoma cells;

Fig. 6 affects result figure to lung adenocarcinoma cell multiplication capacity after FoxQ1 gene disturbs;

Fig. 7 is the result of variations figure to the invasive ability of lung adenocarcinoma cell invasion and attack transfer ability after FoxQ1 gene disturbs;

Fig. 8 is the result of variations figure after FoxQ1 gene disturbs, the transfer ability of lung adenocarcinoma cell invasion and attack transfer ability being changed;

Fig. 9 is the cell statistical graph to lung adenocarcinoma cell invasion and attack transfer ability after FoxQ1 gene disturbs;

Figure 10 is the result of variations figure to the apoptosis index of correlation of Apoptosis of Lung Adenocarcinoma Cell ability after FoxQ1 gene disturbs;

Figure 11 is each index band gray-scale value scintigram to Apoptosis of Lung Adenocarcinoma Cell ability after FoxQ1 gene disturbs;

Figure 12 is the streaming apoptosis result to Apoptosis of Lung Adenocarcinoma Cell ability after FoxQ1 gene disturbs;

Figure 13 is the qualification result figure in stable cell strain building process;

Figure 14 be after FoxQ1 gene disturbs, nude mice animal is become to knurl respectively organize tumour picture;

Figure 15 becomes the growth curve chart of knurl to nude mice animal after FoxQ1 gene disturbs;

Figure 16 is that the tumour cell of the effect of the collaborative cis-platinum of siRNA changes result figure to cisplatin chemotherapy susceptibility;

Figure 17 respectively organizes tumour picture after the injection cis-platinum of effect of the collaborative cis-platinum of siRNA;

Figure 18 respectively organizes tumor growth curve after the injection cis-platinum of effect of the collaborative cis-platinum of siRNA.

Embodiment

Below in conjunction with specific embodiment, the present invention is described further, but the present invention is embodiment, is not limited to this.

The main raw that following examples are used is: lung cancer cell line 4 strains: SPC-A-1, and A549, HC C827 and NCI-H1395, be purchased from typical case's culture collection council of Chinese Academy of Sciences cell bank.The anti-human Fo xQ1 of rabbit, Bcl-2, Bax, Fas-L monoclonal antibody, mouse-anti people Caspase-3 monoclonal antibody, is purchased from Abcam.Male 4 week age BALB/c nude mice, body weight 18-20g, is purchased from Shanghai Slac Experimental Animal Co., Ltd., nude mice is raised in Nantong University's Experimental Animal Center barrier environment.

The design of the siRNA of embodiment 1 target gene FoxQ1

Retrieval NCBI GeneBank obtains FoxQ1 complete sequence and RNA sequence, utilizes existing network resource and popular software to carry out biological analysis to FoxQ1, selects coding region as the target sequence of siRNA design.On existing siRNA design website, carry out primary design and screening, then with reference to following principle of design, screen.The principle of SiRNA sequences Design: (1), from the AUG initiation codon of mRNA, is found " AA " two and connected sequence, and write down the base sequence of its 3 ' end, as potential siRNA target site; (2) select GC content at the siRNA of 30%-50% than higher more effective of GC content; (3) avoid secondary structure to repeat; (4) 3 ' of sence chain end stability is held low than 5 '; (5) potential sequence and corresponding gene group database are carried out to homologous sequence search, get rid of the sequence with other encoding sequence or EST homology.Get rid of 5 ' of aitisense chain and hold the potential siRNA of continuous 8 montages and the pairing of other gene; The potential siRNA that gets rid of any one section continuous 14 bases and the pairing of other gene.1 pair of siRNA sequence of final acquisition, for:

Positive-sense strand: 5 '-GCCAAGCAAUUUCUUUAAATT-3 ',

Antisense strand: 5 '-UUUAAAGAAAUUGCUUGGCTT-3 '.

Embodiment 2 cell cultures

Cell recovery: take out SPC-A-1, NCI-H1395, HCC827, A549 cell from liquid nitrogen, put into immediately 37 ℃ of water baths and melt fast, cell is thawed completely in 1min.With behind 75% alcohol wipe sterilization cryopreservation tube surface, be transferred in the fresh RPMI-1640 nutrient solution of 10mL and wash, the centrifugal 5min of 1000rpm.Absorb supernatant, get 6mL RPMI1640 perfect medium (RPMI1640 basic medium+10%FBS+1% penicillin and streptomycin) and add centrifuge tube, mix, be transferred in culturing bottle, put in 37 ℃ 5%CO ₂cell culture incubator in cultivate, change next day after a nutrient solution, continue to cultivate.

Passage is cultivated: the old nutrient solution covering with in the culturing bottle of cell is discarded, PBS rinses twice, add 1mL to contain 0.02%EDTA+0.25% trysinization liquid, digestion 3～5min, observation of cell state under microscope, becomes circle when cell volume dwindles, when gap becomes large, the RPMI-1640 nutrient solution that adds 5mL to contain 10% foetal calf serum stops digestion, at the bottom of repeatedly blowing and beating bottle with suction pipe.By in the new culturing bottle of the cell suspension packing after piping and druming well, then add appropriate nutrient solution, be placed in 37 ℃, 5%CO ₂cell culture incubator in cultivate.

Cell cryopreservation: select the cell of logarithmic phase degree of converging approximately 90%, conventional peptic cell, collecting cell suspension, centrifugal 1000r/min, 5min.Suck supernatant, add frozen storing liquid (DMSO: FBS=1: 9), count, adjusting cell concn is 5 * 10 ⁶/ mL left and right.Be in charge of every pipe 1mL.Cryopreservation tube is obturaged, indicated cell category and frozen date.4 ℃ of 30min successively ,-20 ℃ of 60min ,-80 ℃ of 24h liquid nitrogen gas-bearing formations (approximately-100 ℃) 30min, liquid nitrogen.

Embodiment 3 real-time fluorescence quantitative RT-PCRs and detected by Western blot (Western Blot) detect the expression of FoxQ1 gene and albumen in four strain cells

Extract respectively total RNA and the total protein of four strain cells, by real-time fluorescence quantitative RT-PCR and detected by Western blot (Westem Blot), detect the expression of FoxQ1 gene and albumen in four strain cells.Detailed process is as follows:

1 real-time fluorescence quantitative RT-PCR

(1) total RNA extracts: collect four strain cell strains, to 1.5mL RNA-free centrifuge tube, add 1mL Trizol, fully mix, the standing 5min of room temperature.Add 0.2mL chloroform (trichloromethane), thermal agitation 15s, the standing 2min of room temperature.4 ℃ centrifugal, and 12000r/min * 15min draws supernatant (noting not being drawn onto lower floor) to another 1.5mL centrifuge tube.Add the Virahol with supernatant equivalent, mix gently the standing 10min of room temperature.4 ℃ centrifugal, and 12000r/min * 10min, abandons supernatant.75% ethanol (joining with DEPC water) that adds 1mL precooling, washing precipitation gently, 4 ℃ are centrifugal, and 7500r/min * 5min, abandons supernatant.Dry, be dissolved in 20 μ L DEPC water (65 ℃ of dissolution 10-15min).Uv-spectrophotometric instrument is measured concentration (the μ g/ μ L of unit) and the purity of extracting RNA, with the zeroing of DEPC water, OD ₂₆₀/ OD ₂₈₀between 1.8-2.0, show that the RNA purity of extracting is good.

(2) reverse transcription is cDNA (reverse transcriptase, RT): reverse transcription carries out according to Revert AidT M First Strand cDNA synthesis kit specification sheets.Concrete reaction system is as follows:

Mix rear centrifugally, hatch 60min for 42 ℃, 70 ℃ of 5min of termination reaction.-20 ℃ save backup.

(3) fluorescence quantitative RT-RCR: detailed process is carried out with reference to Maxima SYBR Green/ROX qPCR Master Kit test kit, reacts as follows:

With Applied Biosystems7500Real Time PCR System instrument, react.After 95 ℃ of 10min denaturations, enter PCR circulation.95 ℃ of 15s sex change, 60 ℃ of 60s annealing are extended, and 40 circulations are carried out in reaction.

2 detected by Western blot (Western Blot)

(1) extraction of total protein: the cell of collecting logarithmic phase, add appropriate lysate (containing 1% PMSF and inhibitors of phosphatases), concussion cracking 20min on ice after mixing on vibrator, the centrifugal 20min of 12000r/min, get supernatant, be total protein of cell.The concentration of uv-spectrophotometric instrument Detection and Extraction albumen.According to albumen volume: SDS-PAGE albumen sample-loading buffer volume=4: 1 ratio, add albumen sample-loading buffer, 100 ℃ are boiled 10min, and also-80 ℃ of Refrigerator stores of packing are standby.

(2) join polyacrylamide gel: according to the gum concentration of joining of the size configure suitable concn of molecular weight of albumen.By for joining after the sheet glass clean dry of glue, after alignment, to put into folder and clamp, vertical card on the top of the shelf, is prepared encapsulating (firmly clamping preventing from cementing leakage).First add separation gel, first quick and back slow, prevent Bubble formation, while generally adding to apart from 1.5cm place, upper end, add deionized water sealing, after standing 40min, discard ionized water, add concentrated glue to top, insert comb, prepare loading.

(3) loading electrophoresis: all protein samples are adjusted to isoconcentration 30 μ g, every hole loading approximately 15～20 μ l, wherein a hole adds the protein Marker dying in advance.Starting voltage is 80V, about 40min, and after tetrabromophenol sulfonphthalein enters separation gel, voltage changes 100V into, and about 60min stops electrophoresis when tetrabromophenol sulfonphthalein approaches separation gel bottom.

(4) transferring film: first by the pvdf membrane of suitable size as for activating 10min in methyl alcohol, pvdf membrane is being covered on the gel after electrophoresis again, the noncontact face of pvdf membrane and gel all covers filter paper and sponge, is placed in electric turn trough electricity and turns, and Constant Electric Current turns 300mA * 120min.

(5) sealing: after electricity turns, take out pvdf membrane, put into the confining liquid (containing 1 * TBST of 5% skim-milk) of existing preparation, 4 ℃ are spent the night or room temperature is shaken 2h.Notice that film faces up.

(6) incubate primary antibodie: after sealing finishes, take out, put into and contain appropriate 1 * TBST and wash film 5min * 3 time; Preparation is containing the primary antibodie (each dilutes 2mL puts in centrifuge tube for 1000 times of anti-FoxQ1 antibody dilutions, 1000 times of anti-β-actin antibody dilutions) of 5% skim-milk 1 * TBST dilution; Washed film is placed on dull and stereotyped upper, the facing up of film, the even dropping of antibody of dilute is on film; The about 2h of incubated at room, then put into 4 ℃ and spend the night.

(7) wash film, incubate two and resist: take out pvdf membrane, 1 * TBST liquid is washed 3 times, each 10min, limit side washing is shaken.Join two anti-(two 1000 times of the anti-dilutions, dilution 2mL is put in centrifuge tube) containing 1 * TBST dilution of 5% skim-milk; By two anti-evenly the droppings on film of having diluted, incubated at room 2h; 1 * TBST (not containing skim-milk) cleans 10min * 3 time.

(8) develop: press chemical luminescence reagent kit specification sheets and the A of ECL luminescent solution, B liquid equal proportion are mixed to (preparation before using); Washed film is pasted to angle with filter paper to be blotted; Film is faced up and is placed on plastics film, on film, drip A, B mixed solution; Gel imaging system is taken pictures, is preserved.

As Figure 1-5, wherein Fig. 1 is that real-time fluorescence quantitative RT-PCR detects the expression of FoxQ1 gene in four strain cells to result, and Fig. 2 is solubility curve, and Fig. 3 is amplification curve.Fig. 4 and Fig. 5 are that Western Blot detects expression and the gray scale scanning figure of FoxQ1 albumen in four strain cells.Visible FoxQ1 is the highest at SPC-A-1 cells, in NCI-H1395, takes second place, and A549 and HCC827 cells are lower.

Embodiment 4 cell transfectings

(1) transfection the day before yesterday, collect the cell of logarithmic phase, be inoculated in six orifice plates, inoculation quantity is (3.0-8.0) * 10 ⁵, add 2mL nonreactive substratum;

(2) in 250 μ L RPMI1640 basic mediums, add 80nMSiRNA, soft mixing;

(3) with 250 μ L RPMI1640 basic mediums, dilute 5 μ L lipofectamine, blow and beat 3-5 time gently the standing 5min of room temperature;

(4) mix transfection reagent and siRNA diluent, blow gently the standing 20min of room temperature even 3-5 time;

(5) turn then 4-6h and change liquid, continue to cultivate after 24h or 48h, carry out other detecting step after transfection.

Embodiment 5CCK8 method detects transfection front and back cell proliferation

(1) collect logarithmic phase cell (embodiment 1 preparation), regulate cell density, every hole adds approximately 2000 cells, adds 96 orifice plates, every hole 100 μ L;

(2) after cell attachment, use lipofectamine mediation transfection (described in embodiment 4), while getting after transfection 4h, changing the liquid time is CCK8 experiment 0h, detects respectively 0h, 24h, 48h, 72h cell proliferation situation;

(3) every hole adds 10 μ L CCK8 solution, cultivates after 2h absorbancy (A) value by multi-functional microplate reader in 450nm (650nm reference) each hole of wavelength measurement.

Set 3 multiple holes for every group, repeat to test 3 times, get its average.

As shown in Figure 6, after FoxQ1 gene disturbs, its multiplication capacity obviously reduces compared with negative control combination blank group result.

Embodiment 6Transwell cell is observed the change of transfection front and back cell migration, invasive ability

Transwell cell model detects the change of transfer ability: the cell (transfection preparation method is with embodiment 4) of getting experimental group cell, negative control group cell and untransfected after liposome transfection, use respectively after 0.25% tryptic digestion, resuspended with base training after cell counting, adjusting cell density is 2 * 10 ⁵/ mL.In upper chamber, add 100 μ L cell suspensions, cell count is 2 * 10 ⁴individual, lower chamber is the RPMI-1640 nutrient solution 600 μ L containing 20% foetal calf serum, 37 ℃, 5%CO ₂in incubator, hatch 24h.Hatch after end, take out cell, with PBS, wash twice, cotton swab is chamber filter membrane medial surface attached cell in wiping gently, and PBS washes twice.Cell filter membrane is fixed to 10 minutes with 4 % paraformaldehydes, suck stationary liquid, every hole adds 600 μ L Xylene Brilliant Cyanine G dye liquors, and dyeing 5min, inhales and abandon staining fluid, and PBS washes three times, upper chamber is taken out to seasoning.Just putting the cell count of taking pictures under fluorescent microscope and counting the migration of the film back side, middle body and random 3 visuals field of peripheral part (totally 15 visuals field) of counting every film, calculating mean value.

Transwell cell model detects the change of invasive ability: l4 ℃ of the BD matrige that is stored in-20 ℃ of refrigerators spent the night, liquefy, on ice the matrigel having melted is being diluted according to 1: 4 with the substratum of serum-free, mix, then add 50 μ L to Transwell cell, hatch 4h for 37 ℃, treat that gelling is solid.Remaining step is the same.

As Figure 7-9, after FoxQ1 gene silencing, the invasion and attack transfer ability of SPC-A-1 cell obviously reduces compared with negative control combination blank group result, and difference has statistical significance (P < 0.05).

Embodiment 7Western Blot and Flow cytometry are disturbed the change of front and back apoptosis capacity

After transfectional cell, with Western Blot, detect apoptosis-related protein Bcl-2 before and after transfection, Bax, Cas pase, the variation of Fas-L, and by Flow cytometry, respectively organize the difference of apoptosis.Detailed process is as follows:

(1) transfection the day before yesterday, collect the cell (embodiment 1 preparation) of logarithmic phase, be inoculated in six orifice plates, inoculation quantity is (3.0-8.0) * 10 ⁵, add 2mL nonreactive substratum;

(2) cell transfecting (with embodiment 4), after transfection 48h, centrifugal collecting cell, 2000rpm, 5 minutes, abandons substratum;

(3) cold PBS washed twice;

(4) by 400 μ L1 * Binding Buffer suspension cells, concentration 1 * 10 ⁶/ mL;

(5) in suspension cell, add 5 μ L AnnexinV-FITC, mix gently, 4 ℃ of lucifuges are hatched 15min;

(6) mix gently after adding 10mL PI, 4 ℃ of lucifuges are hatched 5min;

In (7) 1 hours, with flow cytometer, detect the apoptosis situation of respectively organizing.

Result is as shown in Figure 10-12, and Figure 10 is that Western Blot detects transfection front and back apoptosis-related protein Bc1-2, Bax, and Caspase, the variation of Fas-L, Figure 11 is the histogram that the gray-scale value of Western Blot band is painted.After FoxQ1 gene disturbs, short apoptogene Caspase-3, Bax, Fas-L express compared with control group and express and increase, and anti-apoptotic genes expression Bcl-2 expresses compared with control group expression minimizing, difference has statistical significance (P < 0.05), the expression indifference (P > 0.05) of each apoptotic proteins between negative control group and untransfected group.

Flow cytometry disturbs the change result of front and back apoptosis capacity as Figure 12, and after FoxQ1 gene silencing, apoptotic cell accounts for 22.8% of total cellular score, and negative control and blank apoptosis cell account for respectively 4.8% and 7.6% of cell count.Interference group is compared with control group, and difference has statistical significance (P < 0.05).Showing to reduce the apoptosis that can promote lung carcinoma cell after the expression of FoxQ1, is consistent with Western Blot detected result.

Embodiment 8 is for shRNA eucaryon plasmid and the strain of G418 screening stable transfected cells of FoxQ1

(1) according to the principle of design of carrier specification sheets, the siRNA fragment of the reticent FoxQ1 gene of specificity is designed to be able to the hair clip siRNA Insert Fragment (as shown in table 1) in insertion vector, then be connected with pGPU6/GFP/Neo carrier, build and obtain pGPU6/GFP/Neo-siRNA expression vector.Carrier carries GFP reporter gene and G418 screening site.

Hair clip siRNA Insert Fragment

siRNA	?
		Positive-sense strand	5′-GCCAAGCAATTTCTTTAAA-3′
Antisense strand	5′-TTTAAAGAAATTGCTTGGC-3′

(2) the cell SPC-A-1 of the high expression level FoxQ1 gene screening is inoculated in to 6 orifice plates by 3*105/number of perforations above, be positioned over 37 ℃, 5%CO2 incubator is cultivated after 24h, with cationic-liposome lipof ectamineTM2000, the expression vector building is carried out to transfection experiment (transfection method is with embodiment 4) above, 4h after transfection, changes fresh medium and continues to cultivate.Turn after 48h cell dissociation, carry out diluted passage (1: 10), different gradients are set and screen suitable G418 screening concentration and suitable cell inoculation quantity.By G418 dosing, filter out the cell strain of stable transfection sh-FoxQ1, it is 200ng/ μ L that G418 maintains concentration, goes down to posterity and amplification cultivation, and whether real-time fluorescence quantitative RT-PCR detects transfection sequence and exist.

As shown in figure 13, the expression of its FoxQ1 gene of the cell strain of stable transfection continues lower than control group result, illustrates that surely turning strain successfully constructs.

The foundation of embodiment 9 lung cancer Nude mice models

(1) cell of Batch Culture stable transfection sh-FoxQ1, the cell of transfection empty carrier and non-transfected cells.

(embodiment 4)

(2) with 0.25% tryptic digestion after-blow, break into individual cells respectively, resuspended standby with base training.

(3) it is subcutaneous that each group is inoculated respectively 1x107/200 μ L to 4-6 week Balb/c nude mice back.

(4) when tumour grows to 1g left and right, carry out interior generation, tumor cell suspension concentration is adjusted into 5-10x107/mL, only, interference group and control group are inoculated into respectively under the flank of nude mice left and right 0.2mL/, establish and repeat contrast.

(5) tumour becomes knurl the 7th day, selects at random 3 nude mices, and abdominal injection cis-platinum (7.5mg/kg) is observed the growth of respectively organizing nude mice tumour.

(6) within every three days, observe, after growing tumour, with length and the width of tape measuring tumour, and calculate gross tumor volume, draw tumor growth curve.

Result is as shown in Figure 14-15, and Figure 14 is that after tumour is taken out, interference group and negative control group tumour are seen substantially, and the tumour of interference group nude mice is obviously dwindled compared with control group phase specific volume, has statistical significance (P < 0.05).As shown in figure 15, interference group tumor growth is obviously slow for tumor growth curve, and difference has statistical significance (P < 0.05).

The effect of the collaborative cis-platinum of embodiment 10siRNA

24h after cell transfecting organizes the cis-platinum that adds different concns in cell at each, adopts CCK-8 method to observe after dosing 48h, the change of SPC-A-1 cell to cisplatin sensitivity.Detailed process is as follows:

(1) collect logarithmic phase cell, regulate cell density, every hole adds approximately 5000 cells, adds 96 orifice plates, every hole 100 μ L.

(2), after cell attachment, use lipofectamine mediation transfection (transfection step is with embodiment 4).

(3) after transfection 24h, add chemotherapeutic cis-platinum, medicine final concentration is respectively 0,0.1,1,10,25,50,100nM.

(4) add after chemotherapeutic 48H to take out cell and by CCK8 method, survey the OD value of corresponding time test holes, step is with embodiment 5.

(5) inhibiting rate=1-under each concentration (recording OD value-blank OD value)/(negative control-blank OD value) * 100%.With the same time point of paired data t inspection statistics, respectively organize inhibiting rate and whether have significant difference.

Result, as Figure 16 figure demonstration, is compared with blank group with negative control group, and transfection group obviously strengthens chemotherapy drug susceptibility, and comparing difference has statistical significance (P < 0.05).

Meanwhile, in animal level, in nude mice tumour, become knurl the 7th day, select at random three nude mices, abdominal injection cis-platinum, observes tumor growth, measures gross tumor volume and weight, as shown in the figure, Figure 17 is that the tumour of simple cis-platinum group and interference+cis-platinum group is seen substantially to result, and Figure 18 is each group tumor growth curve.Result shows, interference group injection cis-platinum group tumour obviously increases the susceptibility of cis-platinum, and nude mice tumour is significantly less than control group, and difference has statistical significance.

Claims (4)

1. target FoxQ1 gene disturbs a siRNA, it is characterized in that: the sequence of described siRNA is as follows:

Positive-sense strand: 5 '-GCCAAGCAAUUUCUUUAAATT-3 ',

Antisense strand: 5 '-UUUAAAGAAAUUGCUUGGCTT-3 '.

2. the target FoxQ1 gene that a clone has the right described in requirement 1 disturbs the carrier of siRNA.

3. the construction process of carrier claimed in claim 2, it is characterized in that, concrete steps are: siRNA sequence is designed to be able to the hair clip siRNA Insert Fragment in insertion vector, the siRNA sequence of design is connected with pGPU6/GFP/Neo carrier, obtains the interference microRNA for anti-nonsmall-cell lung cancer biological behaviour.

4. target FoxQ1 gene according to claim 1 disturbs the application of siRNA in reticent FoxQ1 gene specifically.

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