CN109762821B - Interfering RNA for inhibiting expression of AFAP1-AS1 and application of interfering RNA in increasing sensitivity of breast cancer radiotherapy - Google Patents
Interfering RNA for inhibiting expression of AFAP1-AS1 and application of interfering RNA in increasing sensitivity of breast cancer radiotherapy Download PDFInfo
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Abstract
The invention relates to the technical field of biology, and particularly discloses an interfering RNA for inhibiting expression of AFAP1-AS1 and application thereof in increasing breast cancer radiotherapy sensitivity. Experiments show that the long-chain non-coding RNA (lncRNA) AFAP1-AS1 is highly expressed in the radiation-resistant breast cancer cells, and the interfering RNA provided by the invention can obviously inhibit the expression of lncRNA AFAP1-AS1 in the breast cancer cells, can increase the radiotherapy sensitivity of the breast cancer cells, and has a very positive effect on the treatment of the breast cancer. The invention also discloses a carrier for carrying the interference RNA for inhibiting the expression of AFAP1-AS1, which can help to consume excessive GSH in radiotherapy resistant tumor cells by adopting a reduction-responsive high molecular polymer, break the intracellular redox balance, improve the ratio of intracellular reactive active oxygen and further improve the radiotherapy sensitivity of the tumor cells. The combination of radiotherapy and gene therapy has more obvious effect on treating tumors.
Description
Technical Field
The invention relates to the technical field of biology, in particular to an interfering RNA for inhibiting AFAP1-AS1 expression and application thereof in increasing breast cancer radiotherapy sensitivity.
Background
The incidence of cancer is on the rise year by year and is one of the most serious diseases threatening human health at present, wherein the incidence of breast cancer is on the rise from the end of the 70 th 20 th century. 1 in the lifetime of 8 women in the United states suffers from breast cancer; china is not a high-incidence country of breast cancer, but is not optimistic, and the growth rate of the incidence of breast cancer in China is 1-2% higher than that of the high-incidence country in recent years. According to 2009 breast cancer onset data published by the national cancer center and health department disease prevention and control agency 2012, it is shown that: the incidence of breast cancer of women in the national tumor registration area is 1 st of malignant tumors of women, the incidence (thickness) of breast cancer of women is 42.55/10 ten thousand in total nationwide, 51.91/10 ten thousand in cities and 23.12/10 ten thousand in rural areas.
Surgery, chemotherapy and radiation therapy (radiotherapy for short) are conventional cancer treatments. Almost half of cancer patients receive radiation therapy. Of the cured cancer patients, nearly 40% benefit from radiation therapy. Most cancer patients are very sensitive to radiotherapy at the beginning of treatment, but the patients gradually develop acquired radiotherapy resistance in the treatment process, so that tumor recurrence and invasive metastasis are promoted, and treatment failure is caused. Although increasing the dose of radiation therapy can promote the killing of tumor cells, the radiation inevitably causes serious toxic and side effects to normal cells and tissues.
RNA interference (RNAi) technology has rapidly developed in recent years and can be used for the treatment of various diseases. Compared with small molecule targeted drugs, siRNA has better selectivity on target spots, can specifically bind target genes and down regulate the expression of the target genes, and cannot influence the expression of other normal genes in cells. However, the biggest problem in clinical transformation of RNAi technology is the lack of low-toxicity and high-efficiency carrier for delivering siRNA to lesion sites and cells. For cancer treatment based on RNAi technology, siRNA needs to address a series of physiological barriers during delivery, such as how to target tumor cells, penetrate tumor tissues and cell membranes, efficiently escape from endosomes, and efficiently release siRNA within cytoplasm.
Although the traditional viral vectors such as adenovirus and retrovirus for delivering siRNA can overcome various physiological barriers, the traditional viral vectors have the defects of difficult preparation, small siRNA capacity, poor targeting specificity, strong immunogenicity and the like. The polymer nano material has the advantages of good compatibility, large RNA carrying capacity, low immunogenicity, easy function integration, easy large-scale preparation and the like, and is widely applied to siRNA delivery and tumor treatment. More importantly, compared with normal tissues, the solid tumor tissues have abundant blood vessels, wider vascular wall gaps, poor structural integrity and lymphatic return loss, so that nano-sized particles tend to be enriched and retained in the tumor tissues more easily (namely, the high permeability and retention effect of the tumor tissues, also called EPR effect). Therefore, constructing a novel polymer nano material and using the polymer nano material for efficient transmission of siRNA are always hot research hotspots at home and abroad. Especially in recent years, researchers at home and abroad are dedicated to developing high-molecular nano carrier materials responding to tumor microenvironment and used for high-efficiency siRNA delivery based on the special microenvironment of tumors, such as weak acid and hypoxic environment, specific enzyme overexpression and the like. The tumor microenvironment response nano-carrier reported at present comprises high molecular nano-carriers such as acid response, hypoxic response, enzyme response and the like. These nanocarriers can be stably present in the blood circulation and normal physiological tissues; after reaching the tumor part, the siRNA can quickly respond to the microenvironment inside and outside the tumor cell, accelerate the release efficiency of the siRNA, obviously improve the gene therapy effect and reduce the toxic and side effects.
Glutathione (GSH) is a common reducing substance in the cytoplasm (concentration of about 2-10 × 10)-3M) and extracellular matrix (concentration about 2-10 × 10)-6M) are present in large differences. Therefore, when the reduction-responsive nano material is used for delivering the therapeutic substance, especially the biomacromolecule, the release of the therapeutic substance in cytoplasm can be accelerated, so that the therapeutic effect is improved. Over the past few decades, researchers at home and abroad have reported a variety of polymeric materials for reduction reactions and use in the delivery and treatment of various therapeutic drugs. One representative polymer material is a disulfide bond-containing polymer, which can be rapidly degraded (degradation time is from several minutes to several hours) by a reducing agent (e.g., GSH). This degradation rate is much faster than that of the aliphatic polyester polymer and the polycarbonate nano-material (the degradation process usually lasts for days, weeks or even months). Due to rapid degradation and rapid release in cellsAt present, many researchers at home and abroad are dedicated to constructing a novel nano material with reduction response for efficient delivery of therapeutic drugs, particularly biomacromolecules such as siRNA and tumor treatment. However, the synthesis steps of the reduction-responsive nano materials are complicated, the preparation process is complex, the function is single, and clinical transformation and application cannot be really realized.
Disclosure of Invention
The invention aims to overcome at least one defect (deficiency) of the prior art and provides an interfering RNA for inhibiting expression of AFAP1-AS1, which can inhibit expression of AFAP1-AS1 in breast cancer cells and increase radiotherapy sensitivity of the breast cancer cells.
The technical scheme adopted by the invention is that the interfering RNA for inhibiting expression of AFAP1-AS1 has a sense strand sequence of GCACAGGUUCUCCAAACAATT and is shown AS SEQ NO. 1; the antisense strand sequence is UUGUUUGGAGAACCUGUGCTT, as shown in SEQ NO. 2. Or the sequence of the sense strand of the interfering RNA is GCUACUUCUGUCUCAUUAATT, and is shown as SEQ NO. 3; the antisense strand sequence is UUAAUGAGAC AGAAGUAGCTT, as shown in SEQ NO. 4.
According to the invention, a breast cancer radiation resistant cell strain is established through an experiment, and then the breast cancer radiation resistant cell strain and a parent strain are subjected to high-throughput sequencing, so that the high expression of long-chain non-coding RNA (lncRNA) AFAP1-AS1 in radiation resistant breast cancer cells is found, and the interfering RNA can obviously inhibit the expression of lncRNA AFAP1-AS1 in the breast cancer cells, can increase the radiotherapy sensitivity of the breast cancer cells, and has a very positive effect on the treatment of breast cancer.
The invention provides application of the interfering RNA for inhibiting expression of AFAP1-AS1 in increasing breast cancer radiotherapy sensitivity.
The invention also provides a vector for carrying the interfering RNA for inhibiting the expression of AFAP1-AS 1. In fact, the carrier can use a wide variety of existing materials, and can use high molecular materials such as acid response, hypoxic response, enzyme response and the like.
Preferably, the carrier adopts a reduction-responsive high molecular polymer. The adoption of the reduction-responsive high molecular polymer is beneficial to consuming excessive GSH in radiotherapy resistant tumor cells, breaking intracellular redox balance, improving the ratio of intracellular reactive oxygen, and further improving the radiotherapy sensitivity of the tumor cells.
Preferably, the carrier adopts a high molecular polymer containing disulfide bonds with higher degradation speed, which is helpful for realizing the quick release of the carried substance. Furthermore, the carrier adopts polyester disulfide amide (PDSA), the synthesis method is simple, the mass preparation can be realized by adopting a one-pot method, the conversion is easy, and the application cost is low.
The invention also provides a breast cancer radiotherapy sensitizing drug which comprises the interfering RNA for inhibiting expression of AFAP1-AS 1.
The breast cancer radiotherapy sensitizing drug also comprises the carrier carrying the interfering RNA for inhibiting the expression of AFAP1-AS 1.
Further, the sensitizing drug is a nanoparticle formed by the carrier entrapping the interfering RNA, and the particle size of the nanoparticle is 10-200 nm.
Compared with the prior art, the invention has the beneficial effects that:
(1) experiments show that the long-chain non-coding RNA (lncRNA) AFAP1-AS1 is highly expressed in radiation-resistant breast cancer cells and can be used AS a target for treating the radiation resistance of the breast cancer cells. The invention provides an interfering RNA for inhibiting expression of AFAP1-AS1, which can increase the radiotherapy sensitivity of breast cancer cells by inhibiting expression of lncRNA AFAP1-AS1 in the breast cancer cells, and has very positive effect on the treatment of the breast cancer.
(2) The invention also provides a carrier for carrying the interference RNA for inhibiting the expression of AFAP1-AS1, which can help consume excessive GSH in radiotherapy resistant tumor cells by adopting a reduction-responsive high-molecular polymer, break the intracellular redox balance, improve the ratio of intracellular reactive active oxygen and further improve the radiotherapy sensitivity of the tumor cells.
(3) The invention also provides a breast cancer radiotherapy sensitizing drug, interference RNA is entrapped by the carrier, and expression of AFAP1-AS1 in breast cancer cells can be remarkably inhibited when the breast cancer cells are transfected; the sensitizing medicament is combined with radiotherapy, and has more obvious effect on treating breast cancer cells.
Drawings
FIG. 1 is a graph showing the cell clonality of NC group, si1 group and si2 group after different doses of radiotherapy.
FIG. 2 is a graph of the cell survival scores of NC, si1, and si2 groups after various doses of radiation therapy.
FIG. 3 shows the apoptosis rates of NC group, si1 group and si2 group after different doses of radiotherapy.
FIG. 4 is a statistical chart of the apoptosis rates of NC group, si1 group and si2 group after different doses of radiotherapy.
FIG. 5 is a flow chart of the cell cycle of NC group, si1 group and si2 group after different doses of radiotherapy (the second peak in the flow chart is stage G2).
FIG. 6 shows the cell cycle distribution of NC, si1 and si2 groups after different doses of radiotherapy.
FIG. 7 is a graph showing the change of the tumor formation volume with time in shCTL group, shAFAP1-AS1-1 group and shAFAP1-AS 1-2 group mice without radiotherapy.
FIG. 8 is a graph showing the change of the tumor formation volume with time in shCTL group, shAFAP1-AS1-1 group and shAFAP1-AS 1-2 group mice in radiotherapy.
FIG. 9 shows immunohistochemical results of shCTL group, shAFAP1-AS-1-1 group and shAFAP1-AS-1-2 group mice.
FIG. 10 shows that the MDA-MB-231 cells were treated with different doses of radiotherapy 24, 48, 72 hours later and tested for GSH expression by PCR.
FIG. 11 shows the GSH/GSSH ratio of MDA-MB-231 cells after 24 hours of radiotherapy with different doses.
FIG. 12 is the effect of the difference in concentration of si AFAP1-AS1 carried by PDSA NPs on the knock-down efficiency of AFAP1-AS 1.
FIG. 13 is a graph showing the dose survival curves of the siNC group, the transfected nanomaterial PDSA NPs group, the transfected siNC-carrying PDSA NPs group, the transfected siAFAP1-AS1 group, and the transfected siAFAP1-AS 1-carrying PDSA NPs group after different doses of radiotherapy.
FIG. 14 is a flow chart of apoptosis after different doses of radiotherapy for the siNC group, the PDSA NPs group transfected with nanomaterials, the PDSA NPs group transfected with siNC, the siAFAP1-AS1 group transfected with siAFAP1-AS 1.
FIG. 15 is a statistical chart of the apoptosis rate of siNC group, transfected nanomaterial PDSA NPs group, transfected siNC-carrying PDSANPs group, transfected siAFAP1-AS1 group, and transfected siAFAP1-AS 1-carrying PDSA NPs group after different doses of radiotherapy.
FIG. 16 is a cell cycle flow chart of the siNC group, the PDSA NPs group transfected with nanomaterials, the PDSANPs group transfected with siNC, the siAFAP1-AS1 group transfected with siAFAP1-AS1 after different doses of radiotherapy (the second peak in the flow chart is stage G2).
FIG. 17 shows the cell cycle distribution of siNC group, transfected nanomaterial PDSA NPs group, transfected siNC-carrying PDSANPs group, transfected siAFAP1-AS1 group, and transfected siAFAP1-AS 1-carrying PDSA NPs group after different doses of radiotherapy.
FIG. 18 is a photograph of fluorescence from mice injected tail vein with PDSA NPs carrying the fluorescent marker si AFAP1-AS 1.
FIG. 19 is a graph showing the distribution of nanoparticles on tumors and other organs after tail vein injection of PDSA NPs carrying the fluorescent marker si AFAP1-AS1 into mice.
FIG. 20 is a graph of tumor volume over time for 6 groups of mice.
FIG. 21 shows the organ-related indices and the histopathological sections of 6 groups of mice.
Detailed Description
The present invention will be further described with reference to specific embodiments, but the present invention is not limited to the following embodiments.
Examples
This example provides two interfering RNAs (siAFAP1-AS1) that inhibit expression of AFAP1-AS 1-siAFAP 1-AS1-1 and siAFAP1-AS 1-2. Wherein the gene sequence of siAFAP1-AS1-1 is AS follows: the sense strand is GCACAGGUUCUCCA AACAATT (SEQ No. 1); the antisense strand is UUGUUUGGAGAACCUGUGCTT (SEQ No. 2); the gene sequence of siAFAP1-AS1-2 is: the sense strand is GCUACUUCUGUCU CAUUAATT (SEQ No. 3); the antisense strand sequence is UUAAUGAGACAGAAGUAGCTT (SEQ No. 4). The two interfering RNAs for inhibiting expression of AFAP1-AS1 can obviously inhibit expression of lncRNA AFAP1-AS1 in breast cancer cells, can increase radiotherapy sensitivity of the breast cancer cells, and has very positive effect on treatment of the breast cancer.
A breast cancer radiotherapy sensitizing drug comprises the interfering RNA (si AFAP1-AS1) for inhibiting expression of AFAP1-AS1 and a vector carrying the si AFAP1-AS 1. The sensitizing drug is nanoparticles formed by the carrier encapsulating the interfering RNA, and the particle size of the nanoparticles is 10-200 nm.
In this embodiment, the carrier is polyester disulfide amide (PDSA), and the specific synthesis method is as follows: under the condition of an ice salt bath, Cystine dimethyl ester hydrochloride (Cystine dimethyl ester dihydrate) is dropwise added into a mixed solution of fatty diacid (all fatty diacid such as succinic acid, glutaric acid, adipic acid, pimelic acid, suberic acid, azelaic acid, sebacic acid, undecanedioic acid, dodecanedioic acid and tridecanedioic acid) and triethylamine (the solvent can be a strong polar solvent such as dimethyl sulfoxide, dimethylformamide and the like). After stirring the reaction at room temperature for 15 minutes to 4 hours, the precipitate in the reaction system was removed, the filtrate was concentrated, and the product was precipitated using ethyl acetate. Dissolving the crude product in strong polar solvent such as dimethyl sulfoxide and dimethylformamide, repeatedly precipitating in ethyl acetate for three times, and vacuum drying to obtain purified PDSA. The synthetic route is as follows:
the preparation method of the sensitization medicine nano-particle comprises the following steps:
nanoparticles loaded with siRNA were prepared using a nanoprecipitation method. Selecting dimethylformamide as a solvent, and respectively preparing PDSA and polyethylene glycol modified phospholipid (PEG-lipid) solutions. And then mixing the PDSA solution, the PEG-lipid solution, the siRNA aqueous solution and the cationic liposome, and dropwise adding the mixture into deionized water under the stirring condition. The nanosol solution was then transferred to an ultrafiltration membrane (EMD Millipore, MWCO 100K), the nanoparticles were separated by centrifugation, washed twice with 1mL of deionized water, collected and dispersed into 1mL of PBS buffer solution for future use. The particle size of the obtained nanoparticles is 10-200 nm.
(1) Single-click multi-target model verification that silencing AFAP1-AS1 can restore radiosensitivity of triple-negative breast cancer
In MDA-MB-231 cells, compared to the untreated group (siNC group), the cell clonogenic rates (fig. 1) and cell survival scores (fig. 2) were significantly reduced for each irradiation dose for both the transfected siAFAP1-AS1-1 group (si1 group) and the transfected siAFAP1-AS1-2 group (si2 group), the differences were statistically significant (P < 0.05) for the si1 group and the si2 group, respectively, compared to the siNC group, and a one-click multi-target model was constructed to fit the dose survival curve (see fig. 2). As can be seen from the model, the dose survival curves of si1 and si2 groups are significantly shifted down compared to the siNC group, demonstrating that cell survival and radiation resistance are significantly reduced in both si1 and si2 groups compared to the siNC group. Cell survival scores (SF2), average lethal dose (D0) and quayside dose (Dq) after 2Gy irradiation are calculated after a single-click multi-target model is fitted to a dose survival curve, and the results show that SF2, D0 and Dq values of the si1 group and the si2 group are all obviously reduced compared with the sinC group, and the results prove that the radiation sensitivity of the si1 group and the si2 group is stronger and the difference reaches obvious statistical significance (P is less than 0.01) by comparing the si1 group and the si2 group with the sinC group respectively (see Table 1). The results all prove that after the expression of AFAP1-AS1 is silenced, the radiosensitivity of the triple negative breast cancer cell MDA-MB-231 is obviously recovered, so that the high expression of AFAP1-AS1 can increase the radioresistance of the triple negative breast cancer cell.
TABLE 1
(2) After expression of AFAP1-AS1 is silenced, apoptosis of triple-negative breast cancer cells is increased after radiotherapy
Transfecting siAFAP1-AS1-1 and siAFAP1-AS1-2 into MDA-MB-231 cells, carrying out radiotherapy 24 hours after the cells are attached to the wall, wherein the radiotherapy doses are 0, 6 and 10Gy respectively, treating the cells according to an Annexin V method after the radiotherapy is carried out for 24-48 hours, and then detecting the change of apoptosis by a flow cytometer.
The results of fig. 3 and 4 show that: compared with an untreated group (a siNC group), after the transfection siAFAP1-AS1-1 group (a si1 group) and the transfection siAFAP1-AS1-2 group (a si2 group) are irradiated by the same radiation dose, the apoptosis rate of MDA-MB-231 cells is obviously increased, and the radiation sensitivity is gradually recovered, so that the AFAP1-AS1 can increase the radiation resistance of triple-negative breast cancer cells.
(3) After the expression of AFAP1-AS1 is silenced, the G2 phase block of triple negative breast cancer cells after radiotherapy is more obvious
Transfecting siAFAP1-AS1-1 and siAFAP1-AS1-2 into MDA-MB-231 cells, carrying out radiotherapy 24 hours after the cells are attached to the wall, wherein the radiotherapy doses are 0, 6 and 10Gy respectively, and detecting the change of the cell cycle by a flow cytometer 24-48 hours after the radiotherapy.
The results of fig. 5 and 6 show that: the G2 arrest of MDA-MB-231 cells was more pronounced in the transfected siAFAP1-AS1-1 group (si1 group) and transfected siAFAP1-AS1-2 group (si2 group) after the same radiation dose exposure AS compared to the untreated group (siNC group). Cells are most sensitive to ionizing radiation in preparation for entry into and during mitosis, i.e., they are most sensitive to radiation therapy during the cell cycle when the cells are in the G2/M phase, and thus the cell cycle is arrested in the G2 phase and sensitivity to radiation therapy is significantly increased. Therefore, after silencing expression of AFAP1-AS1, the radiosensitivity of the triple-negative breast cancer cells is gradually restored, thereby proving that AFAP1-AS1 can increase the radioresistance of the triple-negative breast cancer cells.
(4) Effect of AFAP1-AS1 on growth of triple-negative breast cancer nude mouse orthotopic transplantation tumor
This experiment delivers "small interfering RNA" (siRNA) in vivo by cloning the siRNA sequence into a plasmid vector as a "short hairpin RNA" which, when delivered to an animal, is expressed to form a "double stranded RNA" (dsRNA) that is processed by the RNAi pathway.
The lentiviral interference vector used in the experiment is constructed by Guangzhou Egypti biotechnology, Inc., firstly, an shRNA interference fragment is designed aiming at lncRNA AFAP1-AS1, and the interference fragment is inserted into a lentiviral vector pLKO-Tet-ON. The key function of pLKO-Tet-ON vector is that tetracycline induces RNA interference, and when tetracycline is absent, the expression of shRNA is inhibited by TetR protein. When tetracycline is added to the medium, the expression of the shRNA causes the target gene to be knocked down, so that the plasmid needs to be acted under the action of tetracycline. In addition, this plasmid has puromycin resistance gene Puro and can be selected using puromycin. Finally, the constructed lentivirus-mediated shRNA is pLKO-Tet-On-AFAP1-AS1-914 (shRNA 1 for short), pLKO-Tet-On-AFAP1-AS1-1547 (shRNA 2 for short) and pLKO-Tet-On-NC (shRNA NC for short) respectively.
The experiment was divided into 6 groups of 5 nude mice each. 2 groups of the breast cancer orthotopic transplantation tumor models are established by a stable transformation plant line mammary gland fat pad injection method constructed by transfection lentivirus shRNA NC, after the construction is successful, 1 group is not specially treated, and 1 group is subjected to radiotherapy (abbreviated as shCTL group); 2 groups of stable transformants expressed by the shRNA1 silent lncRNA AFAP1-AS1 are used for establishing a breast cancer orthotopic transplantation tumor model, after the construction is successful, 1 group is not specially treated, and 1 group is irradiated (abbreviated AS shAFAP1-AS-1-1 group); 2 groups establish breast cancer orthotopic transplantation tumor models by using stable transformants expressed by the shRNA2 silent lncRNA AFAP1-AS1, 1 group is not specially treated after successful construction, and 1 group carries out radiotherapy (abbreviated AS shAFAP1-AS-1-2 group). 30 nude mice inoculated with three stable transformants all had tumors, and the long diameter and the short diameter of the nude mice orthotopic transplantation tumor were measured periodically by a vernier caliper, with the tumor formation time being 10 days on average. After the transplanted tumor grows out, drawing a growth curve according to the tumor volume until the tumor volume reaches 150cm3In time, the nude mice are fed with the prepared tetracycline without radiotherapy; in the radiotherapy group, except that a nude mouse is fed with prepared tetracycline, radiotherapy is carried out, the radiotherapy scheme is that tumor transplantation in situ is carried out for 10Gy by single irradiation, the mouse is treated by neck breaking 30 days after the mouse dies naturally or becomes tumor, and the tumor is taken and weighed.
The results are shown in FIG. 7 and FIG. 8, and show that the tumor volume of mice is significantly smaller than that of the untreated group after the lncRNA AFAP1-AS1 is silenced in the group without radiotherapy; in the radiotherapy group, after the lncRNA AFAP1-AS1 is silenced, the tumor volume of the mice is obviously smaller than that of the untreated group, and the difference is more obvious than that of the mice in the group without the radiotherapy. Thus, AFAP1-AS1 can increase the tumor forming capability of triple negative breast cancer, and after the expression of AFAP1-AS1 is silenced in a radiotherapy group, the tumor volume of a mouse is obviously smaller than that of an untreated group, and the difference is more obvious than that of a radiotherapy group, so that AFAP1-AS1 can increase the radiation resistance of a mouse in-situ transplantation tumor.
(5) Expression of related protein in immunohistochemical detection of triple negative breast cancer nude mouse orthotopic transplantation tumor
Three specimens are randomly selected from paraffin-embedded three-negative breast cancer nude mouse orthotopic transplantation tumor specimens of each group, and are respectively subjected to immunohistochemical experiments. FIG. 9 shows that after the expression of lncRNA AFAP1-AS1 is silenced, the expression of a proliferation related index ki67 is obviously reduced, the expression of an apoptosis related index Caspase-3 is obviously increased, the expression of a radiosensitivity related index gamma H2AX is obviously increased, and the expression of beta-catenin is obviously reduced. Therefore, after the lncRNA AFAP1-AS1 is silenced, the proliferation of triple negative breast cancer is reduced, the apoptosis is increased, the radiosensitivity is increased, the expression of beta-catenin is reduced, and the Wnt pathway is inhibited. Therefore, the AFAP1-AS1 high expression can promote the proliferation of triple negative breast cancer, reduce the apoptosis of triple negative breast cancer cells, enhance the radiation resistance of triple negative breast cancer, and activate the Wnt pathway by beta-catenin.
In vivo and in vitro studies prove that the high expression of AFAP1-AS1 can promote the proliferation of triple negative breast cancer, reduce the apoptosis of triple negative breast cancer cells and increase the radiation resistance of triple negative breast cancer.
(6) Oxidation-reduction equilibrium detection after radiotherapy in triple-negative breast cancer cell line
PCR experiments are carried out to detect the expression level of GSH 24, 48 and 72 hours after MDA-MB-231 cells are subjected to radiotherapy, and the results show that the expression level of the GSH is correspondingly increased along with time increment and dose increment (see figure 10).
The GSH/GSSH ratio was measured 24 hours after MDA-MB-231 cell radiotherapy and showed a corresponding increase in GSH/GSSH ratio with increasing dose, but this difference was clearly reduced after the addition of PDSA NPs (see FIG. 11).
The experimental results show that the MDA-MB-231 cells are gradually increased along with the time and the dose after radiotherapy, the GSH and the GSH/GSSH ratio are obviously increased, so that the radiosensitivity of the triple negative breast cancer cells is gradually reduced along with the time and the dose, the PDSA NPs are added after the radiotherapy of the triple negative breast cancer cells, the detected GSH/GSSH ratio is not obviously changed along with the increasing of the dose, and the GSH is consumed by the PDSA NPs, so that the radiosensitivity of the triple negative breast cancer cells is gradually recovered.
(7) Efficiency of knocking down genes related to radiotherapeutic resistance by nano particles
PDSA NPs nanoparticles carrying si AFAP1-AS1 were diluted to carry different concentrations of siRNA (0-50nM) and co-cultured with MDA-MB-231 cells for 48 hours, and RNA extraction was performed in PCR experiments to confirm the knockdown efficiency of PDSA NPs carrying different concentrations of si AFAP1-AS 1. The results suggest that in triple negative breast cancer cells MDA-MB-231, the knocking efficiency of AFAP1-AS1 is increased gradually with the gradually increased concentration of PDSA NPs carrying si AFAP1-AS1, and then the concentration of PDSA NPs carrying si AFAP1-AS 150 nM is selected for the subsequent experiments to continue (see FIG. 12).
(8) Single-click multi-target model confirmed that si AFAP1-AS 1-carrying PDSA NPs can increase radiosensitivity of triple-negative breast cancer cells
The triple-negative breast cancer cell MDA-MB-231 cells are divided into 5 groups, 1 group transfects siNC, 1 group transfects nano-material PDSA NPs, 1 group transfects PDSA NPs carrying siNC, 1 group transfects siAFAP1-AS1, 1 group transfects PDSA NPs carrying siAFAP1-AS1, and the method for constructing the single-click multi-target model is the same AS the experiment (1).
The results indicate that compared with the transfected siNC group, the transfected nanomaterial PDSA NPs group and the transfected siNC-carrying PDSA NPs group, the cell clone formation rate and cell survival score of the transfected siAFAP1-AS1 group and the transfected siAFAP1-AS 1-carrying PDSA NPs group are both obviously reduced, and the transfected siAFAP1-AS 1-carrying PDSA NPs group is more obvious, and the difference reaches the statistical significance (P < 0.05). A one-click multi-target model was constructed, dose survival curves were fitted, and SF2, D0, Dq values were calculated. It can be seen from the model that the dose survival curves of the transfected siAFAP1-AS1 group and the PDSA NPs carrying siAFAP1-AS1 are obviously shifted downwards, the SF2, D0 and Dq values are all obviously reduced, the dose survival curve of the PDSA NPs carrying siAFAP1-AS1 is reduced more obviously, and the SF2, D0 and Dq values are reduced more obviously. The cell survival rate and the radiosensitivity after the PDSA NPs group cell radiotherapy carrying the si AFAP1-AS1 are lower. It can therefore be demonstrated that the nanomaterials PDSA NPs and si AFAP1-AS1 can synergistically increase the radiosensitivity of triple negative breast cancer (see fig. 13, table 2).
TABLE 2
The remarks are as follows: p1 is a comparison of the set of sAFAP 1-AS1 with the set of PDSA NPs carrying the siNC, and P2 is a comparison of the set of PDSA NPs carrying the sAFAP 1-AS1 with the set of PDSA NPs carrying the siNC
(9) Apoptosis experiments prove that the PDSA NPs carrying si AFAP1-AS1 can increase the radiosensitivity of triple-negative breast cancer cells
The triple negative breast cancer cell MDA-MB-231 cells are divided into 5 groups, 1 group transfects siNC, 1 group transfects nano material PDSA NPs, 1 group transfects PDSA NPs carrying siNC, 1 group transfects si AFAP1-AS1, and 1 group transfects PDSA NPs carrying si AFAP1-AS 1. And (3) carrying out radiotherapy 24 hours after the cells are attached to the wall, wherein the radiotherapy doses are 0, 6 and 10Gy respectively, treating the cells according to an Annexin V method 24-48 hours after the radiotherapy, and then detecting the change of apoptosis by using a flow cytometer.
The results show that compared with the transfected siNC group, the transfected nanomaterial PDSA NPs group and the transfected siNC-carrying PDSANPs group, the apoptosis rate of the transfected siAFAP1-AS1 group and the transfected siAFAP1-AS 1-carrying PDSA NPs group is obviously increased, and the transfected siAFAP1-AS 1-carrying PDSA NPs group is more obvious, so that the group of cells are proved to have more apoptosis after radiotherapy and higher sensitivity to radiation. It can thus be demonstrated that the nanomaterials PDSA NPs and si AFAP1-AS1 can synergistically increase the radiosensitivity of triple negative breast cancer (see fig. 14 and 15).
(10) Cell cycle experiments prove that the PDSA NPs carrying si AFAP1-AS1 can increase the radiosensitivity of triple-negative breast cancer cells
The triple negative breast cancer MDA-MB-231 cells are divided into 5 groups, 1 group transfects siNC, 1 group transfects nano-material PDSA NPs, 1 group transfects PDSA NPs carrying siNC, 1 group transfects siAFAP1-AS1, and 1 group transfects PDSA NPs carrying siAFAP1-AS 1. And (3) carrying out radiotherapy 24 hours after the cells are attached to the wall, wherein the radiotherapy doses are 0, 6 and 10Gy respectively, and detecting the change of the cell cycle by a flow cytometer 24-48 hours after the radiotherapy.
The results show that compared with the transfected siNC group, the transfected nanomaterial PDSA NPs group and the transfected siNC-carrying PDSANPs group, the cell cycle detection of the transfected siAFAP1-AS1 group and the transfected siAFAP1-AS 1-carrying PDSA NPs group is obviously G2 cycle arrest, and the cell sensitivity of the group of cells to radiation is proved to be higher by the fact that the transfected siAFAP1-AS 1-carrying PDSA NPs group is more obvious. It can thus be demonstrated that the nanomaterials PDSA NPs and si AFAP1-AS1 can synergistically increase the radiosensitivity of triple negative breast cancer (see fig. 16 and 17).
The early in vitro and in vivo studies prove that the high expression of AFAP1-AS1 can promote the proliferation and the metastasis of triple negative breast cancer and increase the radiation resistance of the triple negative breast cancer. Therefore, AFAP1-AS1 may likely be a therapeutic target for triple negative breast cancer. In-vivo research in the experiment proves that the novel nano-material PDSA NPs carrying si AFAP1-AS1 can increase the radiosensitivity of triple negative breast cancer cells, and the effect is more obvious than that of using the si AFAP1-AS1 alone.
(11) Evaluation of tumor enrichment Capacity of nanoparticles
And inoculating the triple-negative breast cancer MDA-MB-231 cells to the subcutaneous part of a female nude mouse to construct a subcutaneous tumor model. When the tumor volume reaches 100mm3And injecting PDSA NPs carrying the fluorescence labeling si AFAP1-AS1 into a mouse body through tail vein, and observing the distribution of the nanoparticles in the mouse body and the tumor enrichment condition by using a small animal living body imager. The results in fig. 18 and 19 show that the tumor tissue enrichment of PDSA NPs loaded with si AFAP1-AS1 is significantly higher than that of the rat tail vein PDSA NPs group and PBS group.
(12) In vivo tumor growth inhibiting effect of nanoparticles
The method for establishing breast cancer orthotopic transplantation tumor model by injecting triple negative breast cancer cells MDA-MB-231 row breast fat pad into 30 female BALB/C nude miceAnd (4) molding. The long diameter and the short diameter of the nude mice orthotopic transplantation tumor are regularly measured by a vernier caliper, and the tumor formation time is 10 days on average. After the in-situ transplanted tumor grows out, drawing a growth curve according to the tumor volume until the tumor volume reaches 150cm2 Dividing 30 tumor-bearing mice into 6 groups, each group comprises 5 mice, wherein two groups of mice are injected with PBS through rat tail veins, and then one group of mice is taken for radiotherapy; injecting PDSA NPs nanometer materials into the two groups of rat tail veins, and then taking one group of the rat tail veins for radiotherapy; two groups of rat tail intravenous PDSA NPs material carried si AFAP1-AS1, one of which was then taken for radiotherapy. The radiotherapy program is to perform 10Gy of single irradiation on the tumor transplanted in situ. And (3) after the mice die naturally or become tumor, the neck of the mice is cut off 30 days, and the tumor is taken and weighed.
The results show that tumor volumes of six groups of mice are weighed and compared in volume, and according to the results of growth curves, the tumor volumes of the six groups of mice are reduced most obviously particularly in mice of a rat tail vein injection group of PDSA NPs material carrying si AFAP1-AS1 and a group of PDSA NPs material carrying si AFAP1-AS1 combined with radiotherapy. In vivo studies have thus demonstrated that AFAP1-AS1 in combination with PDSA NPs treatment can restore the radiosensitivity of triple negative breast cancer, and therefore triple negative breast cancer can be effectively treated by combination with PDSA NPs material carrying si AFAP1-AS 1-l in combination with radiotherapy (see FIG. 20).
(13) In vivo toxicity testing of nanoparticles
PDSA NPs loaded with si AFAP1-AS1 were injected tail vein into mice (n ═ 3) at a dose of 1nmol siRNA per mouse. Injecting once a day, killing the mice after continuously injecting for 3 days, collecting peripheral blood of the mice, and carrying out blood routine detection; at the same time, different organs were collected and fixed with paraformaldehyde. Sections were then sectioned and H & E stained for histological analysis. The results in FIG. 21 show that the PDSA NPs carrying siAFAP1-AS1 have no significant toxic side effects in mice.
It should be understood that the above-mentioned embodiments of the present invention are only examples for clearly illustrating the technical solutions of the present invention, and are not intended to limit the specific embodiments of the present invention. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention claims should be included in the protection scope of the present invention claims.
Claims (5)
1. Use of an interfering RNA that inhibits expression of AFAP1-AS1 to increase breast cancer radiation therapy sensitivity in the manufacture of a medicament for increasing breast cancer radiation therapy sensitivity, wherein the interfering RNA has a sense strand sequence of GCACAGGUUCUCCAAACAATT; the antisense strand sequence is UUGUUUGGAGAACCUGUGCTT.
2. Use of a vector in the preparation of a medicament for increasing breast cancer radiation therapy sensitivity, wherein the vector carries an interfering RNA that inhibits expression of AFAP1-AS1 to increase breast cancer radiation therapy sensitivity, and the sense strand sequence of the interfering RNA is GCACAGGUUCUCCAAACAATT; the antisense strand sequence is UUGUUUGGAGAACCUGUGCTT.
3. Use according to claim 2, wherein the support is a reduction-responsive high molecular polymer.
4. Use according to claim 3, wherein the carrier is a disulfide bond-containing high molecular weight polymer.
5. The use according to any one of claims 2 to 4, wherein the medicament is a nanoparticle formed by the interfering RNA entrapped in the carrier, and the particle size of the nanoparticle is 10 to 200 nm.
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