CN110038025B

CN110038025B - Preparation method of RNA triple-helix hydrogel for targeted therapy of triple-negative breast cancer

Info

Publication number: CN110038025B
Application number: CN201910346420.8A
Authority: CN
Inventors: 李雪梅; 丁来荣; 赵飞浪; 张书圣
Original assignee: Linyi University
Current assignee: Linyi University
Priority date: 2019-04-26
Filing date: 2019-04-26
Publication date: 2021-08-17
Anticipated expiration: 2039-04-26
Also published as: CN110038025A

Abstract

The invention discloses a preparation method of RNA triple-helix hydrogel for targeted therapy of triple-negative breast cancer. The vector is a pure RNA system and is formed in a rolling circle transcription mode, a therapeutic gene CXCR4 is generated in the transcription and replication process, and the other two therapeutic microRNAs, namely a tumor-inhibiting microRNA and an oncomiR inhibitor, are hybridized; a complementary sequence of the aptamer capable of targeting MDA-MB-231 cells is designed on a linear template, cholesterol is designed on the complementary sequence of the aptamer, and an RNA hydrogel is formed after high-speed centrifugation. The RNA triple-helix hydrogel is successfully applied to a drug sustained-release system, has good biocompatibility, can simultaneously deliver three therapeutic genes due to the addition of the triple-helix structure, and has wide prospects in the fields of growth inhibition of targeting cells MDA-MB-231 tumor cells, biomedicine and the like.

Description

Preparation method of RNA triple-helix hydrogel for targeted therapy of triple-negative breast cancer

Technical Field

The invention relates to the technical field of biochemical nano materials, in particular to a preparation method of RNA triple-helix hydrogel for targeted therapy of triple-negative breast cancer.

Background

Breast cancer is one of the most common cancers in women, Triple Negative Breast Cancer (TNBCs) refers to a type of breast cancer that lacks expression of Estrogen Receptor (ER), Progesterone Receptor (PR), and human epidermal growth factor receptor 2(HER 2). Clinical studies have demonstrated that TNBCs are the most aggressive type of breast cancer than other types of breast cancer, with poor prognosis, and a more difficult to control mortality and metastatic propensity. Traditional therapies include hormone therapy, targeting three receptors, but the therapeutic effect is less than satisfactory. Therefore, more effective development of TNBCs diagnostics and combination therapies is urgent and important.

The chemokine receptor therapeutic gene CXCR4 has been shown to be one of the key factors in human cancer metastasis, CXCR4 plays an important role in the migration of hematopoietic progenitor/stem cells through interaction with ligands, stromal cell derived factor receptor, and the infiltration and metastasis of breast cancer cells are successfully locked in animal models by the successful prevention of chemokine receptor CXCR4 gene expression with RNA.

miRNA-221 located on intron 6 of Trpm1 gene is a tumor suppressor factor for controlling expression of Cyclin D1 and CDK6, and messenger RNA of microRNA-221 stimulates and draws MDA-MB-231 and SKBr3 breast cancer cells and tumor microenvironment. microRNA-205 plays an important role in triple negative breast cancer by regulating the targeted genes E2F1 and LAMC1 that reduce cell proliferation in vivo and in vitro.

In view of the above existing problems and the advantages of pure RNA hydrogels, there is a need to develop a new hydrogel that is only self-assembled by RNA molecules and is highly effective in treating triple negative breast cancer and a preparation method thereof.

Disclosure of Invention

The technical problem to be solved by the invention is as follows: the preparation method of the hydrogel which is only self-assembled by RNA molecules and has the advantages of high efficiency, low toxicity and side effects, good biocompatibility and the like and is used for efficiently treating the triple negative breast cancer is provided.

The technical scheme of the invention is as follows:

the preparation method of the RNA triple-helix hydrogel for the targeted therapy of triple negative breast cancer comprises the steps of firstly designing a linear DNA transcription template, forming the hydrogel of a pure RNA system in a rolling circle transcription mode, designing an antisense sequence of a CXCR4 sequence in the linear DNA and a triple-helix antisense sequence which can carry two therapeutic genes at the back, generating a therapeutic gene CXCR4 in the transcription and replication process, then hybridizing the other two therapeutic microRNAs and a nucleic acid aptamer targeting MDA-MB-231 cell by a Watton-Crick and Hoogsteen base complementary pairing principle, and centrifuging to form the RNA triple-helix hydrogel nanostructure.

Further, the nucleotide sequence of the linear DNA transcription template is shown as SEQ ID No.1, and the 5' end is phosphorylated.

Further, the two additional therapeutic micrornas are tumor-suppressor micrornas and oncomiR inhibitors.

Further, the nucleotide sequences of the other two therapeutic microRNAs are shown as SEQ ID No.2 and SEQ ID No.3, and the 5' ends of the nucleotide sequences have FAM groups.

Furthermore, the aptamer targeting the MDA-MB-231 cell is Fam-LXLapt-DNA-Chol which is a nucleotide sequence shown in SEQ ID No.4, the 5 'end of the aptamer is modified with a Fam group, and the 3' end of the aptamer is modified with cholesterol.

The preparation method of the RNA triple-helix hydrogel for targeted therapy of triple negative breast cancer comprises the following specific steps:

(1) taking a linear DNA template which is shown in SEQ ID No.1 and is phosphorylated at the 5' end and carrying out annealing treatment on the linear DNA template and a T7 promoter at the same concentration;

(2) adding T4 ligase and T4 ligase buffer solution, and maintaining the temperature at 16 ℃ for 12h to form an RNA transcription template;

(3) adding T7 polymerase, rNTP, T7 polymerase buffer solution and TEM buffer solution, and maintaining at 37 ℃ for 4h to form a multi-copy RNA hydrogel carrier;

(4) and (3) maintaining the RNA hydrogel carrier obtained in the step (3), the TEM buffer solution, the two pieces of therapeutic microRNA and the aptamer targeting MDA-MB-231 cells at 65 ℃ for 5min, slowly cooling to 25 ℃, placing in a refrigerator at 4 ℃ for 2h, and centrifuging at 10000rpm to form the final RNA triple-helix hydrogel.

Further, the annealing treatment method comprises the following steps: heating to 95 deg.C in TEM buffer solution for 5min, cooling to 25 deg.C at 1 deg.C/min, and maintaining for 30 min.

Further, the TEM buffer solution consists of: 1mM spermine, 10.8mM MgCl₂，10mM Tris-HCl，pH＝8.0。

The RNA triple-helix hydrogel is applied to the preparation of a triple-negative breast cancer targeted therapy medicament.

The aptamer was selected from the group of subjects of Yangyongensis university, Yangyongensis, by screening. And the drug specifically binds with MDA-MB-231 cell surface receptor, thereby realizing targeted drug delivery. The RNA gene nanoprobe constructed as a control group in subsequent experimental work also has a certain targeted therapy effect.

Cholesterol (Chol), which has hydrophobicity, can promote the transcription copies of RNA to form nano-structures and also enhances the stability of the structures.

The hydrogel is a high molecular polymer with three-dimensional network structure and hydrophilicity, the invention adopts supermolecule hydrogel, the hydrogel is directly constructed by RNA, and the formation of the hydrogel is realized by RNA self-assembly technology and high-speed centrifugation. The hydrogel is accurately assembled by the Watton-Crick and Hoogsteen base complementary pairing principle, is short in time consumption and good in biocompatibility, and has wide prospects in the fields of growth inhibition of targeting cell MDA-MB-231 tumor cells, biomedicine and the like.

Compared with the prior art, the invention has the following beneficial effects:

the RNA triple-helix hydrogel disclosed by the invention is successfully applied to a drug sustained release system, has good biocompatibility, can simultaneously deliver three therapeutic genes due to the addition of the triple-helix structure, and has a wide prospect in the fields of growth inhibition of targeting cell MDA-MB-231 tumor cells, biomedicine and the like.

Drawings

FIG. 1 is a schematic diagram of the application of RNA triple-helix hydrogel in tumor cell detection and treatment;

FIG. 2 is an optimized graph of 1% agarose gel electrophoresis on the dosage of microRNA with different concentrations and verifies the formation of macromolecular hydrogel.

FIG. 3 is a transmission electron microscope and scanning electron microscope characterization of triple helix hydrogels hybridized with only three RNA transcript copies of therapeutic genes;

FIG. 4 is a scanning electron microscopy characterization of triple-helical hydrogels hybridized with RNA transcript copies of only three therapeutic genes incubated with 10% FBS at 37 ℃ for 12 h;

FIG. 5 is a representation of a transmission electron microscope and a scanning electron microscope of a Fam-LXL Apt-CholRNA triple helix hydrogel simultaneously modified with hybridization of RNA transcript copies of three therapeutic genes;

FIG. 6 is a scanning electron microscopy characterization of a simultaneously modified Fam-LXLapt-CholRNA triple helix hydrogel LXLapt-DNA-CholRNA triple helix hydrogel hybridized with RNA transcript of three therapeutic genes incubated with 10% FBS at 37 ℃ for 12 h;

FIG. 7 is Zeta potential diagram and particle size diagram of hydrogel at different stages;

FIG. 8 is a CLSM assay and flow cytometric analysis of nano-hydrogel particle uptake by MDA-MB-231 cells targeted to non-targeted cells Hela and MCF-7 cells; a is the position of taking the RNA triple-helix hydrogel by the cell photographed by a confocal microscope; b is a confocal imaging image obtained by applying the same processing method to Hela cells; c is a confocal imaging image obtained by applying the same processing method to MCF-7 cells;

FIG. 9 is a flow cytogram targeting MDA-MB-231 cells for uptake of RNA triple-helical nano-hydrogel particles;

FIG. 10 is a flow cytogram of the uptake of RNA triple-helical nanohydrogel particles by non-targeted cell Hela;

FIG. 11 is a flow cytogram of non-targeted cell MCF-7 versus RNA triple-helical nanohydrogel particle uptake;

FIG. 12 is a graph showing the detection of cytotoxicity;

FIG. 13 is one of three comparative treatments of mice;

FIG. 14 is a graph comparing the effect of treatment on mice in three groups;

FIG. 15 shows the morphology difference of tumor cells and the proliferation of the cells;

FIG. 16 monitoring the change in body weight of nude mice;

FIG. 17 metastasis of cancer cells from the internal organs of three groups of mice;

FIG. 18H @ E staining analysis of all mouse organs.

Detailed Description

The nucleotide sequence of the linear DNA transcription template is shown in SEQ ID No.1, and the 5' end is phosphorylated.

The other two therapeutic microRNAs are tumor-inhibiting microRNAs and oncomiR inhibitors. Preferably microRNA-205 and microRNA-221. The aptamer targeting the MDA-MB-231 cell is Fam-LXLapt-DNA-Chol which is a nucleotide sequence shown in SEQ ID No.4, the 5 'end of the aptamer is modified with a Fam group, and the 3' end of the aptamer is modified with cholesterol.

The RNA nano hydrogel comprises three nano drug delivery carriers of microRNA, and meanwhile, the RNA nano hydrogel carrier can independently carry CXCR4 and non-treatment type microRNA to serve as a control group for subsequent work.

Placing the RNA nano hydrogel and cultured MDA-MB-231 cells into an incubator for incubation under the culture environment condition of 37 ℃ and 5% CO₂Concentration, culturing and incubating for a certain time, and carrying out related detection.

The nano probe RNA hydrogel acts on the body of a mouse, a TEM buffer and Scrambled RNA with any sequence are used as a control group for experiment, and the treatment effect is observed by monitoring the change tissue change of the tumor volume in the mouse.

The RNA nano hydrogel is simply characterized by a Transmission Electron Microscope (TEM), and then the treatment effect of the RNA nano hydrogel is tested by adopting a laser confocal imaging (CLSM) method and a CCK-8 cytotoxicity method, and the growth inhibition effect of the nano probe on the targeting cell MDA-MB-231 tumor cell is verified by flow cytometry and the like.

The carrier for targeted therapy of triple negative breast cancer, the preparation method and the application provided by the invention utilize the RNA nano hydrogel with gene regulation and control function as the carrier, and have good biocompatibility. The nanometer probe has the growth inhibition effect on the targeting cell MDA-MB-231 tumor cell, and has great application potential in the related research and development of the tumor treatment field.

The oligonucleotide sequences used in this experiment are shown in Table 1

Oligonucleotide sequences used in Table 1

1. The preparation process of the RNA nano hydrogel comprises the following steps:

(1) annealing ssDNA templates in Table 1 with a T7 promoter at the same concentration;

(4) and (3) maintaining the RNA hydrogel carrier obtained in the step (3), a TEM buffer solution, two pieces of therapeutic microRNA (miR-205 antisense and miR-221 antisense in Table 1) and a nucleic acid aptamer targeting MDA-MB-231 cells (Fam-LXL-DNA-Chol in Table 1) at 65 ℃ for 5min, slowly cooling to 25 ℃, placing in a refrigerator at 4 ℃ for 2h, and performing high-speed centrifugation to form the final RNA triple-helix hydrogel.

In order to stabilize the triple helix structure, a large amount of linking of nitrogen-rich compounds is required in the buffer solution, and the TEM buffer solution consists of: 1mM spermine, 10.8mM MgCl₂，10mM Tris-HCl，pH＝8.0。

According to the steps shown in the schematic diagram, firstly, the RNA nano hydrogel containing three miRNAs is successfully prepared based on the processes of DNA self-assembly, RCT reaction and the like (figure 2), and after a series of reactions, a product with relatively large molecular mass is finally formed along with the change of conditions. As shown in fig. 3, the hydrogel of unmodified cholesterol was morphologically characterized simply by TEM and SEM, and it can be seen that its particle size can reach micron level, in fig. 4 we designed it to incubate in 10% FBS for 12h, and by SEM characterization of its state it can be seen that its morphology has been disrupted and thus is not sufficiently stable in serum during circulation. By using the same method and method, the hydrogel modified with cholesterol is characterized by TEM and SEM, the size of the hydrogel can be controlled to be about 200nm in the electron microscope, the SEM shows that the hydrogel is greatly agglomerated together (figure 5), and when the hydrogel modified with cholesterol is incubated with 10% FBS for 12h and is characterized by SEM (figure 6), the shape of the hydrogel is complete, so the hydrogel can be stably present in serum in the circulating process. Thus improving the stability of the hydrogel in serum after modification of cholesterol on the aptamer and binding to the hydrogel.

In order to determine the forming property of the hydrogel in different stages, the hydrogel in different stages is subjected to Zeta potential characterization, as shown in FIG. 7, so that the particle size of the hydrogel is controlled due to the addition of cholesterol, the stability of the hydrogel in the body circulation process is improved, and the electronegativity of the hydrogel is even reduced.

2. For the characterization of the RNA triple-helix nano-hydrogel for the targeted uptake of cells, the specific operation process is as follows:

placing MDA-MB-231 cells in a 35mm glass button culture dish, incubating for 24h at 37 ℃, adding RNA triple helix hydrogel when the cell density reaches about 80%, incubating for 2h, adding DAPI cell nucleus stain, calibrating the position of cell nucleus, removing culture solution by PBS, suspending the cells in 1mL PBS, photographing the uptake position of the cells to the RNA triple helix hydrogel by a confocal microscope, as shown in A in FIG. 8, collecting the cells in a 1.5mL centrifuge tube after the completion of the process, and measuring the flow cytometry to obtain the flow cytometry of FIG. 9. The same processing method was applied to Hela and MCF-7 cells to obtain a confocal imaging map of B, C in fig. 8 and flow cytograms of fig. 11 and 12, respectively.

3. The CCK-8 kit is used for cytotoxicity experiments:

first, 100. mu.L of cell suspension was prepared in a 96-well plate, and the plate was incubated at 37 ℃ with 5% CO₂Preculture for 24h in an incubator, adding 10 mu L of different types of drugs to be detected into the culture plate, putting the culture plate in the incubator for incubation for a certain time, discarding the original culture solution, replacing with 100 mu L of new culture solution, adding 10 mu L of CCK-8 solution (taking care that bubbles cannot be generated in the wells, otherwise the OD value reading is influenced) into each well, continuing incubation in the incubator for a proper time, and measuring the absorbance at 450nm by using a microplate reader. The wells with cell suspension added with CCK-8 only and without test substance were selected as control wells, and the culture medium without cells was selected and added with CCK-8 as blank for experiments. Final cell viability% ([ a (medicated) -a (blank)]/[ A (0 dosing) -A (blank)]×100％。

The results are shown in fig. 12, and under the same experimental conditions, the cell survival rate of the RNA triple-helix hydrogel after the action is obviously lower than that of the RNA triple-helix hydrogel and the rolling circle product after the action. Namely, the RNA triple-helix hydrogel has the best effect on the treatment effect of targeting triple-negative breast cancer cells.

4. In vivo assay

Firstly, establishing MDA-MB-231 cell subcutaneous tumor-bearing mouse models, wherein one group is used as a blank group and is injected with a buffer solution (TEM buffer) used in an experiment; one group is set as a control group, RNA triple helix hydrogel medicines with any sequence are injected into tumor; one group is set as an experimental group, RNA triple helix hydrogel medicine is injected into tumor, the injection dosage of each group is 30 mu L per time, and the injection frequency is consistent. After the injection procedure, follow-up observations were made, and fractional tumor volumes were tracked and recorded. After termination of the experiment, the mice were sacrificed by carbon dioxide asphyxiation, and the tumors and the internal organs of the mice were exfoliated.

FIGS. 13 to 18 are graphs of in vivo data analysis of triple negative breast cancer (MDA-MB-231) tumor-bearing mice administered with TEM buffer, random-sequence triple-helix hydrogel and RNA triple-helix hydrogel.

As can be seen from fig. 13 and 14, the treatment effects of the three groups on the mice are clearly compared, and the treatment effects are consistent with the cytotoxicity curves of fig. 12. In order to further analyze the treatment effect of the three hydrogels on the tumor, the tissues fixed by 4% paraformaldehyde are subjected to paraffin embedding, frozen sectioning, H @ E staining and cell proliferation Ki67 to analyze the morphology difference and cell proliferation condition of tumor cells, as can be seen from fig. 15, after the treatment of the RNA triple helix hydrogel, the tumor cells are infiltrated by a large number of inflammatory cells, the tissues are damaged to a certain extent, while the control group has large and large number of cell nuclei, which indicates that the cancer cells divide rapidly and are still in the cell proliferation stage. By monitoring the change of the weight of the nude mouse, as shown in fig. 16, it is effectively evaluated that the RNA triple-helix hydrogel and the TEM buffer used for the hydrogel have good biocompatibility and do not affect the growth of the mouse. In addition to microscopic analysis, macroscopic observation is also carried out, fig. 17, the internal organs of three groups of mice have no obvious cancer cell metastasis condition from appearance, on the basis, H @ E staining analysis is carried out on all the internal organs of the mice in fig. 18, and the microscopic data show that all the internal organs have no obvious metastasis condition, so that the experimental construction is preliminarily considered as a tumor-bearing system of the human triple-negative breast cancer cells, and the experimental construction has some rejection with the mouse source.

In conclusion, the research and design of the RNA triple-helix hydrogel diagnosis and treatment integrated nano detection treatment platform realizes the targeted triple gene therapy on tumor cells. The role of the hydrogel in this experiment is mainly shown in (1) ligation of aptamers targeting MDA-MB-231; (2) the gene therapy of the triple negative breast cancer is realized by the microRNA-205 and the microRNA-221 through a gene substitution method; (3) as a vector to target the therapeutic gene into the tumor cell. The method adopts laser confocal imaging and flow cytometry for detection, analyzes the intake of hydrogel by in vitro triple negative breast cancer cells, verifies the cytotoxicity by CCK8, establishes a nude mouse model, and presents good treatment effect by tracking the weight, the tumor size and the later pathological analysis of the mouse.

The above-mentioned embodiments only express the specific embodiments of the present application, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present application. It should be noted that, for those skilled in the art, without departing from the technical idea of the present application, several changes and modifications can be made, which are all within the protection scope of the present application.

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Claims

1. The preparation method of the RNA triple-helix hydrogel for targeted therapy of triple negative breast cancer is characterized by comprising the following steps: firstly, designing a linear DNA transcription template, forming hydrogel of a pure RNA system in a rolling circle transcription mode, designing an antisense sequence of a CXCR4 sequence and a triple helix antisense sequence carrying two therapeutic genes at the back in the linear DNA, generating a therapeutic gene CXCR4 in the transcription and replication process, hybridizing the other two therapeutic microRNAs and a nucleic acid aptamer targeting MDA-MB-231 cell by a Watton-Crick and Hoogsteen base complementary pairing principle, and centrifuging to form an RNA triple helix hydrogel nanostructure;

the nucleotide sequence of the linear DNA transcription template is shown as SEQ ID No.1, the 5 'end is phosphorylated, the other two therapeutic microRNAs are microRNAs for tumor inhibition and oncomiR inhibitors, the nucleotide sequences are shown as SEQ ID No.2 and SEQ ID No.3, and the 5' ends of the two therapeutic microRNAs are provided with FAM groups; the aptamer targeting the MDA-MB-231 cell is Fam-LXLapt-DNA-Chol which is a nucleotide sequence shown in SEQ ID No.4, the 5 'end of the aptamer is modified with a Fam group, and the 3' end of the aptamer is modified with cholesterol;

the method comprises the following specific steps:

2. The method for preparing RNA triple-helix hydrogel for targeted therapy of triple negative breast cancer according to claim 1, wherein: the annealing treatment method comprises the following steps: heating to 95 deg.C in TEM buffer solution for 5min, cooling to 25 deg.C at 1 deg.C/min, and maintaining for 30 min.

3. The method for preparing RNA triple-helix hydrogel for targeted therapy of triple negative breast cancer according to claim 1 or 2, characterized in that: the TEM buffer solution consists of: 1mM spermine, 10.8mM MgCl₂，10 mM Tris-HCl，pH=8.0。

4. Use of the RNA triple-helix hydrogel prepared by the preparation method according to any one of claims 1 to 2 in the preparation of a drug for targeted therapy of triple-negative breast cancer.