CN109762821A - Inhibit the RNA interfering of AFAP1-AS1 expression and increases the application in radiotherapy in breast cancer sensibility - Google Patents
Inhibit the RNA interfering of AFAP1-AS1 expression and increases the application in radiotherapy in breast cancer sensibility Download PDFInfo
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Abstract
The present invention relates to field of biotechnology, specifically disclose a kind of RNA interfering that inhibition AFAP1-AS1 is expressed and the application in increase radiotherapy in breast cancer sensibility.Long-chain non-coding RNA (lncRNA) AFAP1-AS1 high expression in the breast cancer cell that radiation is resisted is found through experiments that in the present invention; RNA interfering provided by the present invention can significantly inhibit expression of the lncRNA AFAP1-AS1 in breast cancer cell; the radiation sensitivity that breast cancer cell can be increased has very positive effect for the treatment of breast cancer.The invention also discloses a kind of for carrying the carrier of the above-mentioned RNA interfering for inhibiting AFAP1-AS1 expression, it can facilitate to consume excessive GSH in radiotherapy resistance tumour cell by using the high molecular polymer of reduction response, break redox equilibrium intracellular, the ratio for improving intramicellar reaction active oxygen, further increases the radiation sensitivity of tumour cell.Combined radiotherapy and gene therapy become apparent from the therapeutic effect of tumour.
Description
Technical field
The present invention relates to field of biotechnology, and in particular to it is a kind of inhibit AFAP1-AS1 expression RNA interfering and increasing
Application in radiotherapy in breast cancer sensibility.
Background technique
Cancer morbidity has become one of the disease for threatening human health most serious in trend is risen year by year at present, wherein
Global breast cancer incidence is in rising trend always since the late 1970s.8, U.S. women just has 1 people in life
It suffers from breast cancer；It is also unsuitable optimistic although China is not the high-incidence country of breast cancer, the growth of China's breast cancer incidence in recent years
Speed is but higher by high-incidence national 1~2 percentage point.It is announced within 2012 according to National Cancer Center and prevention and control of diseases office, the Ministry of Public Health
Pathogenesis of breast carcinoma data in 2009 show: national tumour registration area breast cancer incidence occupies the 1st of female malignant
Position, female mammary gland cancer morbidity (rough and careless) whole nation add up to 42.55/10 ten thousand, and city is 51.91/10 ten thousand, rural area 23.12/10
Ten thousand.
Operation, chemotherapy and radiotherapy (abbreviation radiotherapy) are conventional cancer treatment methods.Almost half cancer patient
All receive radiotherapy.In the cancer patient of healing, there is nearly 40% patient all to benefit from radiotherapy.Most of cancer patient is treating
At the beginning of it is very sensitive to radiotherapy, but patient gradually generates acquired radiotherapy over the course for the treatment of and resists, and then promotes tumour
Recurrence and invasion transfer, lead to treatment failure.Although the killing to tumour cell can be promoted by increasing Radiotherapy dosimetry, radiate
Line unavoidably generates serious toxic side effect to normal cell and tissue.
RNA interference (RNA interference, RNAi) technology is quickly grown in recent years, can be used for controlling for a variety of diseases
It treats.It is compared with small molecule targeted drug, siRNA can be more preferable to the selectivity of target spot, can specifically bind target gene and lower
Expression of target gene, and will not influence the expression of other normal genes in cell.But RNAi technology is maximum in clinic conversion
Problem is the absence of less toxic efficient carrier and siRNA is transported to lesions position and intracellular.For the cancer based on RNAi technology
Treatment, siRNA need to solve a series of physiologic barriers in transmission process, for example, how targets neoplastic cells, penetrate tumor tissues
Efficiently escape with cell membrane, from endosome and be released effectively siRNA in cytoplasm etc..
Although traditional viral vectors such as adenovirus and retrovirus transmitting siRNA can overcome above-mentioned various physiology screens
Barrier, but have the shortcomings that preparation is difficult, siRNA capacity is small, targeting specific is poor, immunogenicity is strong.Due to high molecular nanometer material
Material has many advantages, such as that compatibility is good, carries RNA capacity greatly, low immunogenicity, is easy to function integration, is easy large scale preparation,
High molecular material is widely used in siRNA transmitting and oncotherapy.Particular, it is important that relative to normal tissue, solid tumor
Blood vessels in tissue is abundant, vascular wall gap is wider, poor structural integrity, lymphatic return missing, enables the particle of nano-scale
It enough tends to be enriched with and be trapped in tumor tissues (i.e. the high-permeability and retention effect of tumor tissues, also known as EPR effect).Cause
This, constructing novel high polymer nano material and the efficient transmitting for siRNA is always domestic and international research hotspot.It is especially close several
Nian Lai, over-expressed based on the special microenvironment of tumour such as weak acid and weary oxygen environment, certain enzyme etc., domestic and international researcher is dedicated to
It develops the high molecular nanometer carrier material of tumor microenvironment response and is efficiently transmitted for siRNA.At present it has been reported that it is swollen
Tumor microenvironment response nano carrier includes the high molecular nanometers carriers such as acid response, the response of weary oxygen, enzyme response.These nano-carrier energy
It is stabilized in blood circulation and normal physiological tissue；After reaching tumor locus, microenvironment inside and outside tumour cell can be done
Quick response out accelerates the release efficiency of siRNA, significantly improves gene therapy effect and mitigates toxic side effect.
Glutathione (Glutathione, GSH) is a kind of common reducing substances, and in cytoplasm, (concentration is about 2
~10 × 10-3M) and in extracellular matrix (concentration is about 2~10 × 10-6M there are greatest differences for concentration).Therefore, it uses
Reduction response nano material transmitting therapeutic substance especially large biological molecule when, therapeutic substance can be accelerated and released in endochylema
It puts, to improve therapeutic effect.In the past few decades, domestic and international researcher reports the macromolecule material of a variety of reduction reactions
Material, and it is used for the conveying and treatment of various therapeutic agents.One of representative high molecular material is containing disulfide bond
High molecular polymer, this high molecular material under the action of reducing agent (such as GSH), can with fast degradation (degradation time from
A few minutes to a few houres).This degradation speed is significantly faster than fats polyester polymers and the (degradation of polycarbonate-based nano material
Process typically last for a couple of days, several weeks even the several months).Due to that can discharge rapidly with fast degradation and in the cell entrained
Gene or drug the advantages of, at present lot of domestic and international researcher all be dedicated to building reduction response novel nano-material,
Efficient conveying and oncotherapy for therapeutic agent especially large biological molecule such as siRNA.But these restore receiving for response
Rice materials synthesis complex steps, preparation process is complicated, has a single function, and can not really be able to realize clinical conversion and application.
Summary of the invention
The present invention is directed to overcome at least one defect (deficiency) of the above-mentioned prior art, a kind of inhibition AFAP1-AS1 is provided
The RNA interfering of expression, the RNA interfering can inhibit expression of the AFAP1-AS1 in breast cancer cell, increase putting for breast cancer cell
Treat sensibility.
The technical solution adopted by the present invention is that a kind of RNA interfering for inhibiting AFAP1-AS1 expression, the RNA interfering is just
Adopted chain-ordering is GCACAGGUUCUCCAAACAATT, as shown in SEQ NO.1；Antisense strand sequence is
UUGUUUGGAGAACCUGUGCTT, as shown in SEQ NO.2.Alternatively, the sense strand sequence of the RNA interfering is
GCUACUUCUGUCUCAUUAATT, as shown in SEQ NO.3；Antisense strand sequence is UUAAUGAGAC AGAAGUAGCTT, such as SEQ
Shown in NO.4.
The present invention resists cell strain by the radiation of experimental construction breast cancer, then it is carried out high-flux sequence with parent plant,
It expresses it was found that long-chain non-coding RNA (lncRNA) AFAP1-AS1 is high in the breast cancer cell that radiation is resisted, is interfered in the present invention
RNA can significantly inhibit expression of the lncRNA AFAP1-AS1 in breast cancer cell, can increase the radiotherapy of breast cancer cell
Sensibility has very positive effect for the treatment of breast cancer.
The present invention provides the RNA interferings of above-mentioned inhibition AFAP1-AS1 expression to increase radiotherapy of Breast Cancer sensibility
In application.
The present invention also provides a kind of for carrying the carrier of the above-mentioned RNA interfering for inhibiting AFAP1-AS1 expression.It is practical
On, which can be used existing a variety of materials, can be using high molecular materials such as acid response, the response of weary oxygen, enzyme responses.
Preferably, the high molecular polymer that the carrier is responded using reduction.Using the high molecular polymer of reduction response
Help to consume excessive GSH in radiotherapy resistance tumour cell, breaks redox equilibrium intracellular, improve intramicellar reaction active oxygen
Ratio, further increase the radiation sensitivity of tumour cell.
Preferably, the carrier using degradation speed faster containing the high molecular polymer of disulfide bond, help to realize by
The quick release of loading matter.Further, the carrier using two sulphamide of polyester (poly (disulfide amide),
PDSA), synthetic method is simple, can be prepared on a large scale using " one kettle way ", is easy to convert, application cost is low.
The present invention also provides a kind of radiotherapy of Breast Cancer hypersitization medicines, express including above-mentioned inhibition AFAP1-AS1
RNA interfering.
Above-mentioned radiotherapy of Breast Cancer hypersitization medicine further includes the interference that above-mentioned carrying inhibits AFAP1-AS1 expression
The carrier of RNA.
Further, the hypersitization medicine is the nano particle that the carrier contains that this described RNA interfering is formed, described to receive
The partial size of rice grain is 10~200nm.
Compared with prior art, the invention has the benefit that
(1) breast cancer that long-chain non-coding RNA (lncRNA) AFAP1-AS1 is resisted in radiation is found through experiments that in the present invention
High expression, can be used as the target spot for the treatment of breast cancer cell radiotherapy repellence in cell.The present invention provides a kind of inhibition
The RNA interfering of AFAP1-AS1 expression can be increased by inhibiting expression of the lncRNA AFAP1-AS1 in breast cancer cell
The radiation sensitivity of breast cancer cell has very positive effect for the treatment of breast cancer.
(2) the present invention also provides a kind of for carrying the carrier of the above-mentioned RNA interfering for inhibiting AFAP1-AS1 expression,
It can facilitate to consume excessive GSH in radiotherapy resistance tumour cell by using the high molecular polymer of reduction response, break born of the same parents
Internal oxidition reduction balance, improves the ratio of intramicellar reaction active oxygen, further increases the radiation sensitivity of tumour cell.
(3) the present invention also provides a kind of radiotherapy of Breast Cancer hypersitization medicines, contain RNA interfering by carrier, transfection
When breast cancer cell, expression of the AFAP1-AS1 in breast cancer cell can be significantly inhibited；The hypersitization medicine combined radiotherapy one
It rises, it is more obvious for the effect of breast cancer cell treatment.
Detailed description of the invention
Fig. 1 is the cell clonal formation situation map of NC group, si1 group, si2 group after various dose radiotherapy.
Fig. 2 is NC group, si1 group, the curve graph of the cell survival fraction of si2 group after various dose radiotherapy.
Fig. 3 is the apoptosis rate of NC group, si1 group, si2 group after various dose radiotherapy.
Fig. 4 is the apoptosis rate statistical chart of NC group, si1 group, si2 group after various dose radiotherapy.
Fig. 5 is the cell cycle flow cytometer detection figure (the in streaming figure of NC group, si1 group, si2 group after various dose radiotherapy
Two peaks are the G2 phase).
Fig. 6 is NC group, si1 group, the cell cycle distribution of si2 group after various dose radiotherapy.
When Fig. 7 is non-row radiotherapy, shCTL group, shAFAP1-AS1-1 group and shAFAP1--AS1-2 group mouse at knurl
Accumulate the variation diagram with the time.
When Fig. 8 is row radiotherapy, the tumor formation volume of shCTL group, shAFAP1-AS1-1 group and shAFAP1--AS1-2 group mouse
With the variation diagram of time.
Fig. 9 is the ImmunohistochemistryResults Results of shCTL group, shAFAP1-AS-1-1 group and shAFAP1-AS-1-2 group mouse.
Figure 10 was MDA-MB-231 cell 24 after various dose radiotherapy, 48,72 hours, and row PCR experiment detects GSH's
Expression quantity.
Figure 11 is MDA-MB-231 cell 24 hours after various dose radiotherapy, detects the ratio of GSH/GSSH.
Figure 12 is that the different of the concentration that PDSA NPs carries si AFAP1-AS1 strike inefficient shadow to AFAP1-AS1
It rings.
Figure 13 is after various dose radiotherapy, and siNC group, transfection nano material PDSA NPs group, transfection carry siNC's
The dosage survival that PDSA NPs group, transfection si AFAP1-AS1 group and transfection carry the PDSA NPs group of si AFAP1-AS1 is bent
Line.
Figure 14 is after various dose radiotherapy, and siNC group, transfection nano material PDSA NPs group, transfection carry siNC's
PDSA NPs group, transfects the Apoptosis streaming for carrying the PDSA NPs group of si AFAP1-AS1 at transfection si AFAP1-AS1 group
Detection figure.
Figure 15 is after various dose radiotherapy, and siNC group, transfection nano material PDSA NPs group, transfection carry siNC's
The apoptosis rate for the PDSA NPs group that PDSANPs group, transfection si AFAP1-AS1 group, transfection carry si AFAP1-AS1 counts
Figure.
Figure 16 is after various dose radiotherapy, and siNC group, transfection nano material PDSA NPs group, transfection carry siNC's
The cell cycle streaming for the PDSA NPs group that PDSANPs group, transfection si AFAP1-AS1 group, transfection carry si AFAP1-AS1 is examined
Mapping (second peak is the G2 phase in streaming figure).
Figure 17 is after various dose radiotherapy, and siNC group, transfection nano material PDSA NPs group, transfection carry siNC's
PDSANPs group, transfects the cell cycle distribution for carrying the PDSA NPs group of si AFAP1-AS1 at transfection si AFAP1-AS1 group.
Figure 18 is after being loaded in the PDSA NPs tail vein injection of fluorescent marker si AFAP1-AS1 to Mice Body, and mouse is glimmering
Radiograph.
Figure 19 is nanoparticle after being loaded in the PDSA NPs tail vein injection of fluorescent marker si AFAP1-AS1 to Mice Body
Son is in tumour and other organ distribution maps.
Figure 20 is the gross tumor volume of 6 groups of mouse with the variation diagram of time.
Figure 21 is each visceral relationship index and tissue pathological slice of 6 groups of mouse.
Specific embodiment
The present invention is further elaborated With reference to embodiment, but the present invention is not limited to following embodiment party
Formula.
Embodiment
Present embodiments provide two kinds of RNA interfering (siAFAP1-AS1) --- siAFAP1- for inhibiting AFAP1-AS1 expression
AS1-1 and siAFAP1-AS1-2.Wherein, the gene order of siAFAP1-AS1-1 are as follows: positive-sense strand GCACAGGUUCUCCA
AACAATT(SEQ No.1)；Antisense strand is UUGUUUGGAGAACCUGUGCTT (SEQ No.2)；The base of siAFAP1-AS1-2
Because of sequence are as follows: positive-sense strand is GCUACUUCUGUCU CAUUAATT (SEQ No.3)；Antisense strand sequence is
UUAAUGAGACAGAAGUAGCTT(SEQ No.4).Both inhibit the RNA interfering of AFAP1-AS1 expression that can significantly press down
Expression of the lncRNA AFAP1-AS1 processed in breast cancer cell, can increase the radiation sensitivity of breast cancer cell, for cream
The treatment of gland cancer has very positive effect.
A kind of radiotherapy of Breast Cancer hypersitization medicine, including the above-mentioned RNA interfering (si for inhibiting AFAP1-AS1 expression
AFAP1-AS1) and carry si AFAP1-AS1 carrier.Wherein, the hypersitization medicine is that the carrier contains the interference
The nano particle that RNA is formed, the partial size of nano particle are 10~200nm.
In the present embodiment, which is two sulphamide of polyester (poly (disulfide amide), PDSA), specific to close
At method are as follows: under the condition of ice salt bath, by cystine dimethyl hydrochloride (Cystine dimethyl ester
Dihydrochloride fat diacid (including succinic acid, glutaric acid, adipic acid, pimelic acid, suberic acid, nonyl) are added dropwise to
All fat diacids such as diacid, decanedioic acid, 11 carbon diacid, dodecanedioic acid, hendecane dicarboxylic acid) and triethylamine mixed solution
In (solvent can be the intensive polar solvents such as dimethyl sulfoxide, dimethylformamide).Reaction 15 minutes to 4 hours is stirred at room temperature
Afterwards, sediment in reaction system is removed, filtrate is concentrated, is settled out product using ethyl acetate.Crude product is dissolved in dimethyl sulfoxide, two
The intensive polar solvents such as methylformamide, three times, vacuum drying obtains the PDSA of purifying to repeated precipitation in ethyl acetate.Synthetic thread
Road is as follows:
The preparation method of above-mentioned hypersitization medicine nano particle specifically:
The nanoparticle of siRNA is loaded with using the preparation of nanoprecipitation method.Dimethylformamide is chosen as solvent, is matched respectively
Set PDSA and polyethyleneglycol modified phosphatide (PEG-lipid) solution.Then take PDSA solution, PEG-lipid solution, siRNA
Aqueous solution and cationic-liposome mixing, under agitation, are added dropwise in deionized water.Then nano-solution is turned
It moves in ultrafiltration membrane (EMD Millipore, MWCO 100K), is centrifugated nanoparticle, is washed twice using 1 deionized water
Afterwards, it collects nanoparticle and distributes it to spare in 1mL PBS buffer solution.The partial size of obtained nano particle is 10
~200nm.
(1) one-hit multitarget model verifying silencing AFAP1-AS1 can restore the radiosensitivity of triple negative breast cancer
In MDA-MB-231 cell, compared with untreated fish group (siNC group), transfect siAFAP1-AS1-1 group (si1 group)
And transfection siAFAP1-AS1-2 group (si2 group) is in the Cell colonies assay (Fig. 1) and cell survival fraction of each exposure dose
(Fig. 2) is all significantly reduced, and respectively by si1 group, si2 group compared with siNC group, difference reaches statistical significance (P < 0.05), and
It constructs one-hit multitarget models fitting Dose-survival curve (see Fig. 2).From model as it can be seen that the Dose-survival curve of si1 group and si2 group
It is obviously moved down compared with siNC group, it was demonstrated that si1 group, si2 group and siNC group compare, and cell survival rate and radioresistant are obvious
It reduces.According to calculating the cell survival fraction (SF2) after 2Gy irradiation, average after one-hit multitarget models fitting Dose-survival curve
Lethal dose (D0), quasi-field dosage (Dq), the results show that si1 group, SF2, D0, Dq value of si2 group and the comparison of siNC group are significant
It reduces, respectively by si1 group, si2 group compared with siNC group, it was demonstrated that si1 group, the radiosensitivity of si2 group are stronger, and difference reaches aobvious
It writes statistical significance (P < 0.01) (being shown in Table 1).Result above proves after the expression of silencing AFAP1- AS1 that three is negative newborn
The radiosensitivity of adenocarcinoma cell MDA-MB-231 is obviously restored, to prove that AFAP1- AS1 high expression can increase by three feminine genders
The radioresistant of breast cancer cell.
Table 1
(2) after the expression of silencing AFAP1-AS1, apoptosis increases after triple negative breast cancer cell radiotherapy
SiAFAP1-AS1-1 and siAFAP1-AS1-2 are transfected into MDA-MB-231 cell, 24 after cell is adherent
Hour row radiotherapy, Radiotherapy dosimetry are respectively 0,6,10Gy, handle cell by Annexin V method after 24~48 hours after radiotherapy, so
Pass through the variation of flow cytomery Apoptosis afterwards.
Fig. 3 and Fig. 4 be as the result is shown: compared with untreated fish group (siNC group), transfecting siAFAP1-AS1-1 group (si1 group)
And transfection siAFAP1-AS1-2 group (si2 group), after the irradiation of identical radiological dose, the apoptosis rate of MDA-MB-231 cell is obvious
Increase, radiosensitivity is gradually restored, to prove that AFAP1-AS1 can increase the radiation resistance of triple negative breast cancer cell
Property.
(3) after the expression of silencing AFAP1-AS1, the G2 phase after triple negative breast cancer cell radiotherapy blocks more obvious
SiAFAP1-AS1-1 and siAFAP1-AS1-2 are transfected into MDA-MB-231 cell, 24 after cell is adherent
Hour row radiotherapy, Radiotherapy dosimetry are respectively 0,6,10Gy, pass through flow cytomery cell week after radiotherapy after 24~48 hours
The variation of phase.
Figures 5 and 6 are as the result is shown: compared with untreated fish group (siNC group), transfect siAFAP1-AS1-1 group (si1 group) and
SiAFAP1-AS1-2 group (si2 group) is transfected after the irradiation of identical radiological dose, the G2 phase of MDA-MB-231 cell blocks more bright
It is aobvious.Cell is most sensitive to ionising radiation when being prepared to enter into m period and in m period, i.e., in the cell cycle
In it is most sensitive to radiotherapy when cell is in the G2/M phase, therefore cell-cycle arrest is in the G2 phase, to the quick of radiotherapy
Perception obviously increases.Therefore, after the expression of silencing AFAP1-AS1, the radiosensitivity of triple negative breast cancer cell is gradually restored,
To prove that AFAP1-AS1 can increase the radioresistant of triple negative breast cancer cell.
(4) effect that AFAP1-AS1 grows triple negative breast cancer Orthotopic implantation in nude mice tumor
This experiment conveys method used by " siRNA " (siRNA) in the living body, using siRNA sequence as " short
Hairpin RNA " is cloned into plasmid vector, and when it is fed into the animal body, which is expressed, and forms " a double-strand
RNA " (dsRNA), and handled by the channel RNAi.
Slow virus interference carrier employed in this experiment is the building of Yu Guangzhou Ai Ji Bioisystech Co., Ltd, first needle
ShRNA interference fragment is designed to lncRNA AFAP1-AS1, which is inserted into slow virus carrier pLKO-Tet-ON.
The key function of pLKO-Tet-ON carrier is that tetracycline induces RNA interference, and when lacking tetracycline, the expression of shRNA can quilt
TetR albumen inhibits.After tetracycline is added into culture medium, the expression of shRNA then can cause striking for target gene low, therefore should
Plasmid needs can just work under the action of tetracycline.In addition, the plasmid has puromycin resistance gene Puro, it can be with fast
Purine mycin is screened.The shRNA of the lentivirus mediated finally built is respectively pLKO-Tet-On-AFAP1-AS1-914
(referred to as shRNA 1), pLKO-Tet-On-AFAP1-AS1-1547 (referred to as shRNA 2) and pLKO-Tet-On- NC
(abbreviation shRNA NC).
Experiment is divided into 6 groups, every group of 5 nude mices.Wherein turn plant cream to transfect the steady of slow virus shRNA NC building for 2 groups
Gland fat pad injection method establishes breast cancer orthotopic Transplanted tumor model, refuses specially treated, 1 group of row radiotherapy for 1 group after constructing successfully
(referred to as shCTL group)；2 groups are established breast cancer orthotopic transplanting with the strain that surely turns of shRNA1 silencing lncRNA AFAP1-AS1 expression
Tumor model refuses specially treated, 1 group of row radiotherapy (referred to as shAFAP1-AS-1-1 group) for 1 group after constructing successfully；2 groups with
ShRNA2 silencing lncRNA AFAP1-AS1 expression surely turns strain and establishes breast cancer orthotopic Transplanted tumor model, 1 group after constructing successfully
Not specially treated, 1 group of row radiotherapy (referred to as shAFAP1-AS-1-2 group).Three kinds of inoculation surely turn 30 nude mices of strain at
Tumor passes through the major diameter and minor axis of vernier caliper periodic measurement Orthotopic implantation in nude mice tumor, tumor formation time average out to 10 days.Transplantable tumor is long
After out, growth curve is drawn according to tumor volume, reaches 150cm to gross tumor volume3When, not combination radiotherapy group give nude mice feed match
The tetracycline made；Combination radiotherapy group gives row radiotherapy other than giving nude mice and feeding prepared tetracycline, and Radiation treatment plans are to give
With orthotopic transplantation tumor single fraction irradiation 10Gy, 30 days disconnected necks handle mouse after mouse natural death or tumor formation, and tumor is taken to weigh.
As a result as shown in Figure 7 and Figure 8, the results showed that, in not combination radiotherapy group, the expression of silencing lncRNA AFAP1-AS1
Afterwards, the volume of mouse tumor is significantly less than untreated fish group；After combination radiotherapy group, the expression of silencing lncRNA AFAP1-AS1, mouse
The volume of tumour is significantly less than untreated fish group, and relatively not combination radiotherapy group becomes apparent difference.Thus prove that AFAP1-AS1 can be with
Increase the one-tenth knurl ability of triple negative breast cancer, and after combination radiotherapy group, the expression of silencing AFAP1-AS1, the volume of mouse tumor
It is significantly less than untreated fish group, and relatively not combination radiotherapy group becomes apparent difference, it was demonstrated that AFAP1-AS1 can increase mouse and move in situ
Plant the radioresistant of tumor.
(5) immunohistochemistry detects the expression of GAP-associated protein GAP in triple negative breast cancer Orthotopic implantation in nude mice tumor
From the triple negative breast cancer Orthotopic implantation in nude mice tumor sample of paraffin embedding, every group randomly selects three samples, point
It carry out not immunohistochemical experiment.Fig. 9 the result shows that, after the expression of silencing lncRNA AFAP1-AS1, be proliferated index of correlation ki67
Expression significantly reduce, the expression of apoptosis index of correlation Caspase-3 obviously increases, radiosensitivity index of correlation γ H2AX's
Expression obviously increases, and the expression of β-catenin significantly reduces.It is possible thereby to prove, the expression of silencing lncRNA AFAP1-AS1
Afterwards, the proliferation of triple negative breast cancer is reduced, and apoptosis increases, radiosensitivity increases, and β-catenin expression is reduced, and Wnt is logical
Road is suppressed.Hence it is demonstrated that AFAP1-AS1 high expression can promote triple negative breast cancer to be proliferated, triple negative breast cancer cell is reduced
Apoptosis, and enhance triple negative breast cancer radioresistant, while β-catenin activation activation Wnt access can be made.
Pass through in vivo and in vitro, it was demonstrated that AFAP1-AS1 high expression can promote triple negative breast cancer to be proliferated, and it is negative to reduce three
Apoptosis occurs for breast cancer cell, and increases the radioresistant of triple negative breast cancer.
(6) row redox equilibrium detects after radiotherapy in triple negative breast cancer cell strain
24,48,72 hours after MDA-MB-231 cell radiotherapy, row PCR experiment detects the expression quantity of GSH, the results showed that with
The time be incremented by and dosage escalation, the expression quantity of GSH increase accordingly (see Figure 10).
24 hours after MDA-MB-231 cell radiotherapy, the ratio of GSH/GSSH is detected, the results showed that with dosage escalation,
The ratio of GSH/GSSH is increase accordingly, but after addition PDSA NPs, which is obviously reduced (see Figure 11).
It is above-mentioned the experimental results showed that, MDA-MB-231 cell after radiotherapy with time and dosage escalation, GSH and
GSH/GSSH ratio obviously increases, to show the radiosensitivity of triple negative breast cancer cell passing with time and dosage
Increasing is gradually reduced, but PDSA NPs is added after triple negative breast cancer cell radiotherapy, with dosage escalation, the GSH/GSSH of detection
Ratio shows that GSH is consumed by PDSA NPs, the radiosensitivity of triple negative breast cancer cell is gradually restored then without significant change.
(7) nanoparticle strikes drop and the efficiency of related gene is resisted in radiotherapy
The PDSA NPs nano particle for carrying si AFAP1-AS1 be diluted to carry various concentration siRNA (0-50nM) afterwards with
MDA-MB-231 cell co-cultures 48 hours, extracts RNA row PCR experiment then with clear PDSA NPs and carries various concentration si
AFAP1-AS1's strikes poor efficiency.As a result it prompts in triple negative breast cancer cell MDA-MB-231, as PDSA NPs carries si
The concentration of AFAP1-AS1 is stepped up, and the poor efficiency of striking of AFAP1-AS1 is stepped up, and subsequent experimental selects PDSA NPs to take then
The concentration for carrying si AFAP1-AS1 50nM continues (see Figure 12).
(8) it is thin can to increase triple negative breast cancer by the PDSA NPs that one-hit multitarget model validation carries si AFAP1-AS 1
The radiosensitivity of born of the same parents
Triple negative breast cancer cell MDA-MB-231 cell is divided into 5 groups, 1 group of transfection siNC, 1 group of transfection nano material
PDSA NPs, 1 group of transfection carry the PDSA NPs of siNC, 1 group of transfection si AFAP1-AS1, and 1 group of transfection carries si AFAP1-
The PDSA NPs of AS1 constructs the method for one-hit multitarget model with experiment (1).
As a result it prompts, the PDSA NPs of siNC is carried with transfection siNC group, transfection nano material PDSA NPs group and transfection
Group is compared, transfect si AFAP1-AS1 group and transfection carry si AFAP1-AS1 PDSA NPs group Cell colonies assay and
Cell survival fraction is decreased obviously, and is become apparent from the PDSA NPs group that transfection carries si AFAP1-AS1, and difference reaches system
Meter learns meaning (P < 0.05).One-hit multitarget model, fitted dose survival curve are constructed, and calculates SF2, D0, Dq value.From model
As it can be seen that the Dose-survival curve for transfecting si AFAP1-AS1 group and carrying the PDSA NPs group of si AFAP1-AS1 obviously moves down,
SF2, D0, Dq value obviously lower, and to carry the decline of the Dose-survival curve of the PDSA NPs group of si AFAP1-AS1 more
To be obvious, the reduction of SF2, D0, Dq value is also become apparent.After proof carries the PDSA NPs group cell radiotherapy of si AFAP1-AS1
Cell survival rate it is lower, radiosensitivity is higher.Therefore it can prove that nano material PDSA NPs and si AFAP1-AS1 can
To cooperate with the radiosensitivity for increasing triple negative breast cancer (see Figure 13, table 2).
Table 2
※ annotation: P1 is si AFAP1-AS1 group compared with the PDSA NPs group for carrying siNC, and P2 is to carry si AFAP1-AS1
PDSA NPs group compared with the PDSA NPs group for carrying siNC
(9) it confirms to carry the PDSA NPs of si AFAP1-AS1 with cell apoptosis assay can to increase triple negative breast cancer thin
The radiosensitivity of born of the same parents
Triple negative breast cancer cell MDA-MB-231 cell is divided into 5 groups, 1 group of transfection siNC, 1 group of transfection nano material
PDSA NPs, 1 group of transfection carry the PDSA NPs of siNC, 1 group of transfection si AFAP1-AS1, and 1 group of transfection carries si AFAP1-
The PDSA NPs of AS1.The row radiotherapy in 24 hours after cell is adherent, Radiotherapy dosimetry are respectively 0,6,10Gy, and 24~48 is small after radiotherapy
When after by Annexin V method handle cell, then by the variation of flow cytomery Apoptosis.
The results show that carrying the PDSANPs of siNC with transfection siNC group, transfection nano material PDSA NPs group and transfection
Group is compared, and transfection si AFAP1-AS1 group and the apoptosis rate for transfecting the PDSA NPs group for carrying si AFAP1-AS1 obviously rise
Height, and become apparent with the PDSA NPs group that transfection carries si AFAP1-AS1, it was demonstrated that the cell after this group of cell radiotherapy withers
It dies more, it is higher to the sensibility of radioactive ray.Therefore it can prove that nano material PDSA NPs and si AFAP1-AS1 can be assisted
With the radiosensitivity for increasing triple negative breast cancer (see Figure 14 and 15).
(10) confirm that the PDSA NPs for carrying si AFAP1-AS1 can increase triple negative breast cancer with cell cycle experiment
The radiosensitivity of cell
Triple negative breast cancer MDA-MB-231 cell is divided into 5 groups, 1 group of transfection siNC, 1 group of transfection nano material PDSA
NPs, 1 group of transfection carry the PDSA NPs of siNC, 1 group of transfection si AFAP1-AS1, and 1 group of transfection carries si AFAP1-AS1's
PDSA NPs.The row radiotherapy in 24 hours after cell is adherent, Radiotherapy dosimetry are respectively 0,6,10Gy, are led to after 24-48 hours after radiotherapy
The variation of overflow-type cell instrument detection cell cycle.
The results show that carrying the PDSANPs of siNC with transfection siNC group, transfection nano material PDSA NPs group and transfection
Group is compared, and is transfected si AFAP1-AS1 group and is transfected the cell cycle detection for the PDSA NPs group for carrying si AFAP1-AS1 obviously
It is become apparent in G2 Cycle Arrest, and with the PDSA NPs group that transfection carries siAFAP1-AS1, it was demonstrated that this group of cell is to radiation
The sensibility of line is higher.Therefore it can prove that nano material PDSA NPs and si AFAP1-AS1 can cooperate with the negative cream of increase by three
The radiosensitivity of gland cancer (see Figure 16 and 17).
Early period, in vivo and in vitro proved that AFAP1-AS1 high expression can promote the proliferation of triple negative breast cancer, transfer simultaneously
Increase the radioresistant of triple negative breast cancer.Therefore, AFAP1-AS1 is likely to that the therapeutic target of triple negative breast cancer can be become
Point.This part experiment confirms that novel nano-material PDSA NPs carries si AFAP1-AS1 and can increase by three yin by In vivo study
Property breast cancer cell radiosensitivity, effect relatively become apparent using si AFAP1-AS1 merely.
(11) the tumour accumulation ability assessment of nanoparticle
Triple negative breast cancer MDA-MB-231 cell inoculation is subcutaneous to Female nude mice, construct subcutaneous tumor model.To swollen
Knurl product reaches 100mm3Left and right, will be loaded in the PDSA NPs tail vein injection to Mice Body of fluorescent marker si AFAP1-AS1,
Using small animal living body imager observation nanoparticle in rat kidney tissue and tumour enrichment condition.Figure 18 and 19 results are aobvious
Show, the PDSA NPs for being loaded with si AFAP1-AS1 is apparently higher than caudal vein in tumor tissues enriching quantity and injects PDSA NPs merely
Group and injection PBS group.
(12) inhibit tumor growth effect in nanoparticle body
Triple negative breast cancer cell MDA-MB-231 row mammary fat pad injection method is given to 30 female BAl BIcs/C nude mice
Establish breast cancer orthotopic Transplanted tumor model.30 equal tumor formations of nude mice pass through vernier caliper periodic measurement Orthotopic implantation in nude mice tumor
Major diameter and minor axis, tumor formation time average out to 10 days.After orthotopic transplantation tumor is grown, growth curve is drawn according to tumor volume, to
Gross tumor volume reaches 150cm2When, 30 tumor-bearing mices are divided into 6 groups, every group of 5 mouse, wherein two groups of tail vein injections
Then PBS takes wherein one group of row radiotherapy；Two groups of tail vein injection PDSA NPs nano materials, then take wherein one group of row put
It treats；Two groups of tail vein injection PDSA NPs materials carry si AFAP1-AS1, then take wherein one group of row radiotherapy.Radiation treatment plans
To give orthotopic transplantation tumor single fraction irradiation 10Gy.30 days disconnected necks handle mouse after mouse natural death or tumor formation, and tumor is taken to weigh.
The result shows that the knurl of six groups of mouse is taken to weigh, volume vs are carried out, and according to growth curve as a result, six groups
Mouse especially carries si AFAP1-AS1 group with tail vein injection PDSA NPs material and PDSA NPs material carries si
The tumor mass reduction of AFAP1- AS1 combined radiotherapy group mouse is the most obvious.Thus AFAP1-AS1 is confirmed by In vivo study
Combined PD SA NPs treats the radiosensitivity that can restore triple negative breast cancer, therefore carries si with PDSA NPs material
AFAP1- AS1-l joint radiotherapy can effectively treat triple negative breast cancer (see Figure 20).
(13) the toxicity in vivo test of nanoparticle
It will be loaded in the PDSA NPs tail vein injection to Mice Body of si AFAP1-AS1 (n=3), injection dosage is every
Mouse 1nmol siRNA.Injection is primary daily, after continuous injection 3 days, puts to death mouse, collects mouse peripheral blood, carry out blood routine
Detection；Different organs are collected simultaneously, are fixed using paraformaldehyde.Then slice and H&E dyeing, carry out histologic analysis.Figure 21
The results show that carrying the PDSA NPs of siAFAP1-AS1 for mouse without obvious toxic-side effects.
Obviously, the above embodiment of the present invention is only intended to clearly illustrate technical solution of the present invention example, and
It is not the restriction to a specific embodiment of the invention.It is all made within the spirit and principle of claims of the present invention
Any modifications, equivalent replacements, and improvements etc., should all be included in the scope of protection of the claims of the present invention.
Claims (10)
1. a kind of RNA interfering for inhibiting AFAP1-AS1 expression, which is characterized in that the sense strand sequence of the RNA interfering is
GCACAGGUUCUCCAAACAATT；Antisense strand sequence is UUGUUUGGAGAACCUGUGCTT.
2. a kind of RNA interfering for inhibiting AFAP1-AS1 expression, which is characterized in that the sense strand sequence of the RNA interfering is
GCUACUUCUGUCUCAUUAATT；Antisense strand sequence is UUAAUGAGACAGAAGUAGCTT.
3. the RNA interfering according to claim 1 or 2 for inhibiting AFAP1-AS1 expression is quick in increase radiotherapy of Breast Cancer
Application in perception.
4. a kind of carrier of carrying RNA interfering as claimed in claim 1 or 2 for inhibiting AFAP1-AS1 expression.
5. carrier according to claim 4, which is characterized in that the carrier is the high molecular polymer of reduction response.
6. carrier according to claim 5, which is characterized in that the carrier is the high molecular polymer containing disulfide bond.
7. increasing the application in radiotherapy of Breast Cancer sensibility according to the described in any item carriers of claim 4 to 6.
8. a kind of radiotherapy of Breast Cancer hypersitization medicine, which is characterized in that including inhibition according to claim 1 or 2
The RNA interfering of AFAP1-AS1 expression.
9. radiotherapy of Breast Cancer hypersitization medicine according to claim 8, which is characterized in that further include according to claim
4 to 6 described in any item carriers.
10. radiotherapy of Breast Cancer hypersitization medicine according to claim 9, which is characterized in that the hypersitization medicine is institute
It states carrier and contains the nano particle that this described RNA interfering is formed, the partial size of the nano particle is 10~200nm.
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