CN103614379B - A kind of interference siRNA of target FoxQ1 gene and the purposes of anti-nonsmall-cell lung cancer thereof - Google Patents

Abstract

The invention discloses a kind of target FoxQ1 Gene interfere siRNA and application thereof, the sequence of described siRNA is: positive-sense strand: 5 '-GCCAAGCAAUUUCUUUAAATT-3 ', antisense strand: 5 '-UUUAAAGAAAUUGCUUGGCTT-3 '.The present invention uses fluorescence quantitative RT-RCR, Western Blot, CCK8 method, Transwell Cell modeling, the modern molecular biology techniques such as flow cytometry, successfully construct target FoxQ1 Gene interfere siRNA, by after cell levels and animal level research interference FoxQ1 gene 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 the possibility using FoxQ1 as target treatment lung cancer and the effect of assisting chemotherapeutic treatment tumour, this siRNA being with a wide range of applications in reticent FoxQ1 gene specifically.

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 biomedicine technical 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, 1,350,000 people are about had to be diagnosed as lung cancer in the world every year, in recent years, the death toll that lung cancer causes has exceeded 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 multidisciplinary synthesis Therapeutic mode makes the result for the treatment of of nonsmall-cell lung cancer be enhanced in recent years, due to transfer and the reason such as resistance, current mortality ratio is still very high, total five year survival rate only about 15%.Therefore in the urgent need to seeking new diagnosis index and treatment of strengthening comprehension, exploring new therapeutic strategy and therapy target, becoming lung cancer and study one of field of greatest concern.

The pathologic process that the generation development of tumour is a multistage, multi-step, polygene participate in, relating to the unconventionality expression of the activation of proto-oncogene, the inactivation of cancer suppressor gene and apoptosis-related genes, is a kind of disease of polygene exception in itself.RNA disturbs (RNA interference, RNAi) be increasingly develop the gene silent technology emerging with ripe gradually at present, endogenous or exogenous double-stranded RNA (double stranded RNA, dsRNA) on the post-transcriptional level of gene, in mediated cell there is selective degradation in mRNA, cause expression of target gene reticent, thus 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.Large quantity research shows, RNAi is the ancient phenomenon being extensively present in organic sphere, and different organisms comprises in plant, fruit bat, nematode and some mammalian cells and all there is RNAi phenomenon.RNAi is conservative in organic evolution; that organism resists virus or other external nucleic acid is invaded and keeps the protective mechanism of self inheritance stability; be comprise the mankind from lower eukaryotes to mammalian body in naturally occurring gene silencing mechanism; it suppresses the efficiency of expression of target gene high more than antisense nucleic acid, and the time length is also longer.Under the prerequisite not affecting normal gene function, RNA interference can suppress sudden change or the overexpression of the important gene in some Carcinogenesis, thus reaches gene therapy purpose.Constantly perfect along with RNA perturbation technique, the RNAi for various malignant tumour has achieved the effect of affirmative.The gene therapy research that is found to be of RNAi phenomenon provides strong instrument.SiRNA is to the suppression efficiency of target gene apparently higher than antisense nucleic acid, and compared with antisense nucleic acid, siRNA consumption is lower, and overcomes some shortcomings of antisense nucleic acid, as the non-specific binding of oligomer, easily degraded etc.The siRN of chemosynthesis to proceed to fast in cell and to have an effect, and meanwhile, the siRNA of chemosynthesis costly, easily degrades, only transient expression, and can only continue week age about it.What be unfavorable for further long-term stability carries out study mechanism.

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 and play very important role in some malignant tumour of the mankind develops.In addition, FoxQ1, by transforming growth factor-beta (transforming growth factor-β 1, TGF-β 1) inducible up regulation, participates in epithelial cell form and reinvents and break up.Current research finds that FoxQ1 expresses in intestinal cancer tissue and significantly increases, and the research such as Kaneda shows that FoxQ1 regulates and controls the expression of cancer suppressor gene p21, plays an important role in intestinal cancer generation, development.Zhang etc. report most breast cancer tissue high expression level FoxQ1, and process LAN FoxQ1 in breast cancer cell, can promote cell proliferation and Clone formation in vitro, and strengthen cancer cell invasion power, process LAN FoxQ1 can promote breast cancer cell Lung metastases in vivo.

In the research of lung cancer, seminar in previous experiments confirm and in PLOS ONE magazine ran (PMID:22761930; Doi:10.1371), FoxQ1 is high expression level in most of nonsmall-cell lung cancer (NSCLC) tissue, and with malignancy and patient's prognosis closely related, 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 interference to suppress FoxQ1 in NSCLC, the growth of NSCLC can be suppressed, the biological characteristics of NSCLC is had an impact.

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 FoxQ1 Gene interfere small molecules siRNA, can reticent FoxQ1 gene specifically.Another object of the object of the invention is to provide the carrier containing 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 also can be by expression vector stably express.For the siRNA sequence of FoxQ1, carry siRNA sequence by forms such as liposome, bioabsorbable carrier material, virus vector.The present invention is directed to the coding region sequence of FoxQ1 gene, design a pair siRNA sequence according to corresponding siRNA principle of design, and by transfected with human lung adenocarcinoma cell, have detected the silence efficiency to FoxQ1 gene, confirm its validity and specificity.Then effective siRNA fragment is designed to the hair clip siRNA Insert Fragment inserting pGPU6/GFP/Neo carrier, building can the expression vector of stably express shRNA.

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

A kind of target FoxQ1 Gene interfere siRNA, the sequence of described siRNA is as follows:

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

Antisense strand: 5 '-UUUAAAGAAAUUGCUUGGCTT-3 '.

A kind of clone has the carrier of described target FoxQ1 Gene interfere 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-non-small cell lung Biologic behavior.

The application of described target FoxQ1 Gene interfere 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 modeling, the modern molecular biology techniques such as flow cytometry, successfully construct target FoxQ1 Gene interfere siRNA, by after cell levels and animal level research interference FoxQ1 gene 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 the possibility using FoxQ1 as target treatment lung cancer and the effect of assisting 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 on lung adenocarcinoma cell multiplication capacity after FoxQ1 Gene interfere;

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

Fig. 8 is the result of variations figure to the transfer ability change of lung adenocarcinoma cell invasion and attack transfer ability after FoxQ1 Gene interfere;

Fig. 9 is the Cell counts figure to lung adenocarcinoma cell invasion and attack transfer ability after FoxQ1 Gene interfere;

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

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

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

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

Figure 14 is each group of tumour picture after FoxQ1 Gene interfere, nude mice animal being become to knurl;

Figure 15 is the growth curve chart after FoxQ1 Gene interfere, nude mice animal being become to knurl;

Figure 16 is that siRNA works in coordination with the tumour cell of the effect of cis-platinum to cisplatin chemotherapy susceptibility change result figure;

Figure 17 is each group tumour picture after siRNA works in coordination with the injection cis-platinum of the effect of cis-platinum;

Figure 18 respectively organizes tumor growth curve after siRNA works in coordination with the injection cis-platinum of the effect of cis-platinum.

Embodiment

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

The main raw that following examples use is: lung cancer cell line 4 strain: SPC-A-1, A549, HCC827 and NCI-H1395, is purchased from the American Type Culture Collection council of Chinese Academy of Sciences cell bank.Anti-human FoxQ1, Bcl-2, Bax, Fas-L monoclonal antibody of rabbit, 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., and 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 the target sequence that coding region is designed as siRNA.Design on website at existing siRNA and carry out primary design and screening, then screen with reference to following principle of design.The principle of SiRNA sequences Design: (1), from the AUG initiation codon of mRNA, is found " AA " two and connected sequence, and write down its 3 ' base sequence held, as potential siRNA target site; (2) select GC content more effective more higher than GC content of the siRNA of 30%-50%; (3) secondary structure is avoided to repeat; (4) 3 ' end stability of sence chain is held low than 5 '; (5) potential sequence and corresponding gene group database are carried out homologous sequence search, get rid of the sequence with other encoding sequence or EST homology.Get rid of 5 ' the potential siRNA holding continuous 8 editings and other gene to match of aitisense chain; Get rid of the potential siRNA that any one section continuous 14 bases and other gene match.Final acquisition 1 pair of siRNA sequence, 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, puts into 37 DEG C of water bath fast melt immediately, cell is thawed completely in 1min.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, mixing, is transferred in culturing bottle, puts in 37 DEG C, 5%CO ₂cell culture incubator in cultivate, after changing a nutrient solution next day, continue cultivate.

Passage is cultivated: discarded by the old nutrient solution covered with in the culturing bottle of cell, PBS rinses twice, add 1mL containing 0.02%EDTA+0.25% trysinization liquid, digestion 3 ~ 5min, basis of microscopic observation cell state, becomes circle when cell volume reduces, when gap becomes large, add 5mL and stop digestion, at the bottom of repeatedly blowing and beating bottle with suction pipe containing the RPMI-1640 nutrient solution of 10% foetal calf serum.In the culturing bottle that cell suspension packing after piping and druming is good is new, then add appropriate nutrient solution, be placed in 37 DEG C, 5%CO ₂cell culture incubator in cultivate.

Cell cryopreservation: the cell selecting logarithmic phase degree of converging about 90%, conventional digestion cell, collecting cell suspension, centrifugal 1000r/min, 5min.Suck supernatant, add frozen storing liquid (DMSO: FBS=1: 9), counting, adjustment cell concn is 5 × 10 ⁶about/mL.Be in charge of, often pipe 1mL.Cryopreservation tube is obturaged, indicates cell category and frozen date.4 DEG C of 30min ,-20 DEG C of 60min successively ,-80 DEG C of 24h liquid nitrogen gas-bearing formation (about-100 DEG C) 30min, liquid nitrogen.

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

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

1 real-time fluorescence quantitative RT-PCR

(1) Total RNAs extraction: collect four strain cell strains, in 1.5mL RNA-free centrifuge tube, add 1mL Trizol, fully mix, room temperature leaves standstill 5min.Add 0.2mL chloroform (trichloromethane), thermal agitation 15s, room temperature leaves standstill 2min.4 DEG C centrifugal, 12000r/min × 15min, draws supernatant (noting not being drawn onto lower floor) in another 1.5mL centrifuge tube.Add the Virahol with supernatant equivalent, mix gently, room temperature leaves standstill 10min.4 DEG C centrifugal, and 12000r/min × 10min, abandons supernatant.Add 75% ethanol (joining with DEPC water) of 1mL precooling, washing precipitation gently, 4 DEG C centrifugal, and 7500r/min × 5min, abandons supernatant.Dry, be dissolved in 20 μ L DEPC water (65 DEG C of dissolution 10-15min).UV detector measures the concentration (unit μ g/ μ L) and purity of extracting RNA, with the zeroing of DEPC water, and OD ₂₆₀/ OD ₂₈₀between 1.8-2.0, show that the RNA purity extracted is good.

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

Centrifugal after mixing, hatch 60min for 42 DEG C, termination reaction 70 DEG C of 5min.-20 DEG C save backup.

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

React with Applied Biosystems7500Real Time PCR System instrument.After 95 DEG C of 10min denaturations, enter PCR circulation.95 DEG C of 15s sex change, 60 DEG C of 60s annealing extend, and 40 circulations are carried out in reaction.

2 detected by Western blot (Western Blot)

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

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

(3) loading electrophoresis: all protein samples are adjusted to isoconcentration 30 μ g, every hole loading about 15 ~ 20 μ l, wherein a hole adds the protein Marker of pre-dyed.Starting voltage is 80V, about 40min, and enter after separation gel until tetrabromophenol sulfonphthalein, voltage changes 100V into, about 60min, when tetrabromophenol sulfonphthalein stops electrophoresis close to time bottom separation gel.

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

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

(6) primary antibodie is incubated: close after terminating, take out, put into and wash film 5min × 3 time containing appropriate 1 × TBST; The primary antibodie (each dilution 2mL is put in centrifuge tube for anti-FoxQ1 antibody dilution 1000 times, anti-β-actin antibody dilution 1000 times) that preparation is diluted containing 5% skim-milk 1 × TBST; Be placed on flat board by washed film, facing up of film, the antibody diluted evenly drips on film; Incubated at room is about 2h, then puts into 4 DEG C and spend the night.

(7) wash film, incubate two and resist: take out pvdf membrane, 1 × TBST liquid washes 3 times, each 10min, and limit side washing is shaken.Two anti-(the two anti-dilutions 1000 times, dilution 2mL is put in centrifuge tube) that the 1 × TBST joined containing 5% skim-milk dilutes; Resist diluted two and evenly drip on film, incubated at room 2h; 1 × TBST (not containing skim-milk) cleaning, 10min × 3 time.

(8) develop: A, B liquid equal proportion of ECL luminescent solution is mixed (before using preparation) by chemical luminescence reagent kit specification sheets; Washed film filter paper is pasted angle blot; Being faced up by film puts on a plastic film, and film drips A, B mixed solution; Gel imaging system is taken pictures, preserve.

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 is that Western Blot detects the expression in four strain cells of FoxQ1 albumen and gray scale scanning figure.Visible FoxQ1 is the highest at SPC-A-1 cells, takes second place in NCI-H1395, A549 and HCC827 cells is lower.

Embodiment 4 cell transfecting

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

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

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

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

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

Embodiment 5CCK8 method detects cell proliferation before and after transfection

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

(2) after cell attachment, use lipofectamine mediated transfection (described in embodiment 4), changing the liquid time after getting transfection during 4h is that CCK8 tests 0h, detects 0h, 24h, 48h, 72h cell proliferative conditions respectively;

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

Often organize the multiple hole of setting 3, repeat experiment 3 times, get its average.

Result as shown in Figure 6, after FoxQ1 Gene interfere its multiplication capacity comparatively negative control combination blank group obviously reduce.

Embodiment 6Transwell cell observes the change of cell migration before and after transfection, invasive ability

Transwell Cell modeling detects the change of transfer ability: the cell (transfection preparation method is with embodiment 4) getting the experimental group cell after liposome transfection, negative control group cell and untransfected, after using 0.25% tryptic digestion respectively, resuspended with base training after cell counting, adjustment cell density is 2 × 10 ⁵/ mL.Add 100 μ L cell suspensions in upper room, cell count is 2 × 10 ⁴individual, lower room is the RPMI-1640 nutrient solution 600 μ L containing 20% foetal calf serum, 37 DEG C, 5%CO ₂24h is hatched in incubator.After hatching end, take out cell, wash twice with PBS, cotton swab is room filter membrane medial surface attached cell in wiping gently, and PBS washes twice.Cell filter membrane 4% paraformaldehyde is fixed 10 minutes, sucks stationary liquid, every hole adds 600 μ L Xylene Brilliant Cyanine G dye liquors, and dyeing 5min, inhale and abandon staining fluid, PBS washes three times, is taken out upper room, seasoning.Take pictures under just putting fluorescent microscope and count the cell count of moving at the film back side, counting often opens middle body and random 3 visuals field of peripheral part (totally 15 visuals field) of film, calculating mean value.

Transwell Cell modeling detects the change of invasive ability: spent the night by the BD matrigel4 DEG C being stored in-20 DEG C of refrigerators, liquefy, on ice the substratum of the matrigel serum-free melted is being diluted according to 1: 4, mixing, then 50 μ L to Transwell cells are added, hatch 4h for 37 DEG C, 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 comparatively negative control combination blank group obviously reduces result, and difference has statistical significance (P < 0.05).

The change of embodiment 7Western Blot and Flow cytometry interference front and back apoptosis capacity

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

(1) day before transfection, collects the cell (prepared by embodiment 1) of logarithmic phase, is inoculated in six orifice plates, and 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 washes twice;

(4) with 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 DEG C of lucifuges hatch 15min;

(6) mix gently after adding 10mL PI, 4 DEG C of lucifuges hatch 5min;

Apoptosis situation is respectively organized with flow cytomery in (7) 1 hours.

As shown in figs. 10-12, Figure 10 is that Western Blot detects apoptosis-related protein Bc1-2 before and after transfection, the change of Bax, Caspase, Fas-L, the histogram that the gray-scale value that Figure 11 is Western Blot band is painted to result.After FoxQ1 Gene interfere, short apoptogene Caspase-3, Bax, Fas-L express comparatively control group and express increase, and anti-apoptotic genes expression Bcl-2 expresses comparatively control group expression minimizing, difference has statistical significance (P < 0.05), the expression indifference of each apoptotic proteins between negative control group and untransfected group (P > 0.05).

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

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

(1) according to the principle of design of carrier specification sheets, the siRNA fragment of reticent for specificity FoxQ1 gene 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 screens 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 screened is inoculated in 6 orifice plates by 3*105/number of perforations above, be positioned over 37 DEG C, after 24h cultivated by 5%CO2 incubator, with cationic-liposome lipofectamineTM2000, the expression vector built is carried out transfection experiment (transfection method is with embodiment 4) above, 4h after transfection, changes fresh medium and continues to cultivate.To turn cell dissociation after 48h, carry out diluted passage (1: 10), different gradient is set and screens suitable G418 screening concentration and suitable cell inoculation quantity.Filtered out the cell strain of stable transfection sh-FoxQ1 by G418 dosing, 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 transfected sequences exists.

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 Pulmonary carcinoma nude mice animal model

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

(embodiment 4)

(2) individual cells is broken into 0.25% tryptic digestion after-blow respectively, resuspended for subsequent use with base training.

(3) each group inoculates 1x107/200 μ L to 4-6 week Balb/c nude mice dorsal sc respectively.

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

(5) tumour becomes knurl the 7th day, and Stochastic choice 3 nude mices, abdominal injection cis-platinum (7.5mg/kg), observes the growth of each group of nude mouse tumor.

(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.

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

Embodiment 10siRNA works in coordination with the effect of cis-platinum

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

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

(2), after cell attachment, lipofectamine mediated transfection (transfection procedure is with embodiment 4) is used.

(3) add chemotherapeutic drugs Cisplatin after transfection 24h, medicine final concentration be respectively 0,0.1,1,10,25,50,100nM.

(4) after adding chemotherapeutic, 48H takes out the OD value that cell CCK8 method surveys corresponding time test holes, and step is with embodiment 5.

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

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

Meanwhile, in animal level, become knurl the 7th day at nude mouse tumor, Stochastic choice 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 of tumor growth curve.Result shows, the susceptibility of interference group injection cis-platinum group tumour to cis-platinum obviously increases, and nude mouse tumor is significantly less than control group, and difference has statistical significance.

Claims (4)

1. a target FoxQ1 Gene interfere siRNA, is characterized in that: the sequence of described siRNA is as follows:

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

Antisense strand: 5 '-UUUAAAGAAAUUGCUUGGCTT-3 '.

2. a clone has the right the carrier of the target FoxQ1 Gene interfere siRNA described in requirement 1.

3. the construction process of carrier according to 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-non-small cell lung Biologic behavior.

4. the application of target FoxQ1 Gene interfere siRNA according to claim 1 in the medicine preparing reticent FoxQ1 gene specifically.