CN108743521B - RNA nano hydrogel for targeted therapy of lung cancer and preparation method and application thereof - Google Patents

RNA nano hydrogel for targeted therapy of lung cancer and preparation method and application thereof Download PDF

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CN108743521B
CN108743521B CN201810559190.9A CN201810559190A CN108743521B CN 108743521 B CN108743521 B CN 108743521B CN 201810559190 A CN201810559190 A CN 201810559190A CN 108743521 B CN108743521 B CN 108743521B
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李雪梅
郭英姝
袁丹丹
李俊伟
张书圣
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Abstract

The invention relates to an RNA nano hydrogel for targeted therapy of lung cancer and a preparation method and application thereof, belonging to the technical field of biochemical nano materials. The RNA nano hydrogel for the targeted therapy of the lung cancer provided by the invention comprises a DNA self-assembly single strand, a ring-forming DNA template and a targeted aptamer. The RNA nano hydrogel for the targeted therapy of the lung cancer comprises microRNA which can play a role in gene regulation, can weaken the migration and the invasiveness of lung cancer cells, and can effectively inhibit the lung cancer cells.

Description

RNA nano hydrogel for targeted therapy of lung cancer and preparation method and application thereof
Technical Field
The invention relates to the technical field of biochemical nano materials, in particular to RNA nano hydrogel for targeted therapy of lung cancer and a preparation method and application thereof.
Background
Lung cancer is one of the most common malignancies, with mortality rates residing in the leading prostate of all cancers leading to death. In recent years, the incidence of lung cancer is increasing worldwide, and the health of human beings is seriously threatened. It is already at the forefront to identify new treatment strategies to achieve effective treatment.
For the treatment of lung cancer, the existing treatment technologies mostly adopt surgical treatment and radiotherapy. The operation treatment has the defects of large trauma, high risk, easy generation of complications and the like, and the radiation treatment has larger damage to the normal tissues of the organism.
The microRNA is a short-chain, non-coding and novel gene regulatory factor which is only 17-22 nucleotides and is widely expressed in plants and animals. The existing research shows that microRNA participates in various cell life activities, especially in cancer, the microRNA can be used as an oncogene or a cancer suppressor gene to effectively regulate and control related proteins in cancer cells.
In reports of related technologies at home and abroad, microRNA is used for gene regulation and control, and further gene therapy of cancer is carried out, but most of microRNA plays a role in a single gene and does not have a target recognition effect.
Disclosure of Invention
The invention aims to provide an RNA nano hydrogel for targeted therapy of lung cancer and a preparation method and application thereof. The RNA nano hydrogel for the targeted therapy of the lung cancer comprises microRNA which can play a role in gene regulation, can weaken the migration and the invasiveness of lung cancer cells, and can effectively inhibit the lung cancer cells.
The invention provides an RNA nano hydrogel for targeted therapy of lung cancer, which comprises a DNA self-assembly single strand, a ring-forming DNA template and a targeted aptamer;
the DNA self-assembly single strand comprises ASM-DNA-1 with a nucleotide sequence shown as SEQ ID NO.1, ASM-DNA-2 with a nucleotide sequence shown as SEQ ID NO.2 and ASM-DNA-3 with a nucleotide sequence shown as SEQ ID NO.3, the three DNA single strands respectively take the middle of the sequences as a node, and two ends of the three DNA single strands are in pairwise complementary pairing to form a three-fork self-assembly structure;
the loop-forming DNA template comprises one or more of Three-let7a with a phosphorylated nucleotide sequence at the 5 ' end as shown in SEQ ID NO.4, Three-miR34a with a phosphorylated nucleotide sequence at the 5 ' end as shown in SEQ ID NO.5 and Three-miR 145 with a phosphorylated nucleotide sequence at the 5 ' end as shown in SEQ ID NO.6, and the loop-forming DNA template is complementarily combined with the T7 promoter sequence at the branch end of the Three-fork self-assembly structure through the T7 promoter complementary sequences at the two ends of the sequence;
the targeting aptamer is a substance of which the 5 'end of the sequence of SEQ ID NO.7 is modified with a fluorescent group and the 3' end is modified with cholesterol; the targeting aptamer binds to the circularized DNA template through the T7 promoter complement.
Preferably, the particle size of the RNA nano hydrogel is 80-200 nm.
Preferably, the fluorophore comprises FAM, FITC or Cy 3.
The invention also provides a preparation method of the RNA nano hydrogel in the technical scheme, which comprises the following steps:
1) mixing a DNA self-assembly single strand and a ring-forming DNA template in a Tris-HCl buffer solution for annealing treatment, wherein the annealing treatment conditions are as follows: reducing the temperature to 16-28 ℃ at the speed of 0.5 ℃/min after 5min at 95 ℃ to obtain a three-fork self-assembly structure carrier;
2) mixing the trifurcate self-assembly structure carrier obtained in the step 1) with T4 ligase and T4 ligase buffer solution, and performing cyclization reaction at 16 ℃ to obtain a trifurcate self-assembly structure carrier containing an annular template;
3) mixing the trifurcate self-assembly structure carrier containing the circular template obtained in the step 2) with a nucleotide triphosphate mixed solution, an RNase inhibitor, T7 polymerase and T7 polymerase buffer solution, and carrying out rolling circle transcription reaction at 37 ℃ for 0.5-3 h to obtain a carrier combined with circular DNA;
4) mixing the circular DNA-combined carrier obtained in the step 3) with a targeting aptamer, and carrying out annealing treatment under the conditions of: and (3) reducing the temperature to 16-28 ℃ at the speed of 0.5 ℃/min after 5min at 65 ℃ to obtain the RNA nano hydrogel for the targeted therapy of the lung cancer.
Preferably, the pH value of the Tris-HCl buffer solution in the step 1) is 7.4-8.0.
Preferably, the mixing volume ratio of the DNA self-assembly single strand to the circularized DNA template is 1 (0.8-1.2).
Preferably, the mixing volume ratio of the carrier combined with the circular DNA and the targeting aptamer is 1 (90-110).
The invention also provides application of the RNA nano hydrogel in the technical scheme or the RNA nano hydrogel prepared by the preparation method in the technical scheme in preparation of a medicine for targeted inhibition of lung cancer cells.
Preferably, the lung cancer cells comprise the a549 cell line.
Preferably, the effective dosage of the RNA nano hydrogel in the medicine is 30-150 mu L.
The invention provides an RNA nano hydrogel for targeted therapy of lung cancer. The RNA nano hydrogel for the targeted therapy of the lung cancer comprises a DNA self-assembly single chain, a ring-forming DNA template and a targeted aptamer, wherein the ring-forming DNA template sequence comprises microRNA which plays a role in gene regulation, so that the migration and the invasiveness of lung cancer cells can be weakened, and the lung cancer cells can be effectively inhibited; the combination of the targeting aptamer can ensure that the RNA nano hydrogel has good specific targeting effect, can accurately act on targeted cells, and greatly reduces the damage to other cells; the self-assembly single strand can enable the subsequent three microRNAs to be successfully integrated on the structure of one RNA hydrogel through self-assembly. The specific combination of the DNA self-assembly single-chain, the ring-forming DNA template and the targeting aptamer obtains a drug nano-carrier with great potential, thereby realizing the treatment of the lung cancer. Test results show that the RNA hydrogel can successfully identify targeted cells and play a therapeutic role.
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FIG. 1 is a schematic diagram of RNA nano-hydrogel synthesis provided by the present invention;
FIG. 2 is an electrophoresis characterization diagram and a TEM detection diagram of the RNA nano-hydrogel provided by the invention;
FIG. 3 is a comparison of particle sizes of the RNA nano-hydrogel provided by the present invention before and after the addition of the targeted aptamer modified with cholesterol;
FIG. 4 is a CLSM detection diagram of the RNA nano hydrogel provided by the invention on the target recognition effect of different cells;
FIG. 5 is a flow analysis diagram of the RNA nano-hydrogel provided by the invention for targeting recognition of different cells;
FIG. 6 is a detection diagram of cytotoxicity of the RNA nano hydrogel containing a single ring-forming DNA template, the RNA nano hydrogel containing three ring-forming DNA templates and the hydrogel without microRNA, which are provided by the invention, respectively acting on A549 cells.
Detailed Description
The invention provides RNA nano hydrogel (RNANHs for short) for targeted therapy of lung cancer, which comprises a DNA self-assembly single chain, a ring-forming DNA template and a targeted aptamer;
the DNA self-assembly single strand comprises ASM-DNA-1 with a nucleotide sequence shown as SEQ ID NO.1, ASM-DNA-2 with a nucleotide sequence shown as SEQ ID NO.2 and ASM-DNA-3 with a nucleotide sequence shown as SEQ ID NO.3, the three DNA single strands respectively take the middle of the sequences as a node, and two ends of the three DNA single strands are in pairwise complementary pairing to form a three-fork self-assembly structure;
the loop-forming DNA template comprises one or more of Three-let7a with a phosphorylated nucleotide sequence at the 5 ' end as shown in SEQ ID NO.4, Three-miR34a with a phosphorylated nucleotide sequence at the 5 ' end as shown in SEQ ID NO.5 and Three-miR 145 with a phosphorylated nucleotide sequence at the 5 ' end as shown in SEQ ID NO.6, and the loop-forming DNA template is complementarily combined with the T7 promoter sequence at the branch end of the Three-fork self-assembly structure through the T7 promoter complementary sequences at the two ends of the sequence;
the targeting aptamer is a substance of which the 5 'end of the sequence of SEQ ID NO.7 is modified with a fluorescent group and the 3' end is modified with cholesterol; the targeting aptamer binds to the circularized DNA template through the T7 promoter complement.
In the present invention, the nucleotide sequences involved in the self-assembly of the above-mentioned DNA into a single strand, into a circular DNA template and into a targeting aptamer are shown in Table 1:
TABLE 1 RNA Nanogel related sequences
Figure BDA0001682775730000041
Figure BDA0001682775730000051
In the invention, one end of each of the three DNA self-assembly single-stranded ASM-DNA sequences is provided with a T7 promoter sequence, and the two ends of the circularization DNA template are provided with a complementary sequence of T7, so that the circularization DNA template can be combined with the DNA self-assembly single strand through the complementary sequence. Three cyclization DNA template sequences of three-let7a, three-miR34a and three-miR 145 respectively comprise corresponding microRNA complementary sequences, so corresponding hairpin RNA structures can be respectively generated through a transcription process subsequently, the 5' ends of the three cyclization DNA templates all contain phosphate groups, and the phosphate groups can enable the two ends of a DNA chain to be connected, so that cyclization is facilitated. The targeting aptamer introduces a sequence segment with a lung cancer cell specificity S6 aptamer through base pairing, one end of the sequence is modified with a fluorescent group, the other end of the sequence is modified with cholesterol, and a complementary sequence of a T7 promoter in the targeting aptamer can enable the targeting aptamer to be combined on the transcribed hairpin structure.
The loop-forming DNA template of the RNA nano hydrogel comprises microRNA (let-7a and/or miR34a and/or miR 145) capable of playing a role in gene regulation, wherein the let-7a and miR34a can enable two protooncogenes, namely bcl-2 and c-my (the protooncogenes can inhibit tumor cell apoptosis) to be remarkably reduced, the miR 145 can enable Oct-4 expression to be reduced, and high expression of Oct-4 can also inhibit tumor cell apoptosis, so that the Oct-4 is reduced, and the tumor cell apoptosis is promoted; when multiple microRNAs exist in the RNA nano hydrogel at the same time, the gene therapy effect can be enhanced synergistically, and when three microRNAs exist in the RNA nano hydrogel at the same time, the regulation and control effect of the RNA nano hydrogel is strongest, and the treatment effect is best.
The targeting aptamer is an aptamer of which the sequence contains a sequence of a targeting cell, and can enable the hydrogel to have a targeting recognition effect. In the invention, the addition of the fluorescent group in the targeting aptamer is beneficial to fluorescence imaging observation; in the present invention, the fluorophore includes FAM, FITC or Cy3, and more preferably includes FAM (hydroxyfluorescein) fluorophore. In the invention, the targeted aptamer is modified by cholesterol, so that the RNA nano hydrogel structure can be contracted by utilizing the hydrophobicity of the cholesterol, and the particle size is reduced. In the invention, the particle size of the RNA nano hydrogel is 80-200 nm, and more preferably 150 nm.
The invention also provides a preparation method of the RNA nano hydrogel in the technical scheme, which comprises the following steps:
1) mixing a DNA self-assembly single strand and a ring-forming DNA template in a Tris-HCl buffer solution for annealing treatment, wherein the annealing treatment conditions are as follows: reducing the temperature to 16-28 ℃ at the speed of 0.5 ℃/min after 5min at 95 ℃ to obtain a three-fork self-assembly structure carrier;
2) mixing the trifurcate self-assembly structure carrier obtained in the step 1) with T4 ligase and T4 ligase buffer solution, and performing cyclization reaction at 16 ℃ to obtain a trifurcate self-assembly structure carrier containing an annular template;
3) mixing the trifurcate self-assembly structure carrier containing the circular template obtained in the step 2) with a nucleotide triphosphate mixed solution, an RNase inhibitor, T7 polymerase and T7 polymerase buffer solution, and carrying out rolling circle transcription reaction at 37 ℃ for 0.5-3 h to obtain a carrier combined with circular DNA;
4) mixing the circular DNA-combined carrier obtained in the step 3) with a targeting aptamer, and carrying out annealing treatment under the conditions of: and (3) reducing the temperature to 16-28 ℃ at the speed of 0.5 ℃/min after 5min at 65 ℃ to obtain the RNA nano hydrogel for the targeted therapy of the lung cancer.
The synthesis of the RNA nano hydrogel is realized based on DNA self-assembly and Rolling Circle Transcription (RCT). FIG. 1 is a schematic diagram of RNA nano-hydrogel synthesis provided by the present invention. There is no proportional relationship between the gene segments and the sequences in FIG. 1 of the present invention, and the pictures are only needed for visual representation.
The invention mixes a DNA self-assembly single strand and a ring-forming DNA template in a Tris-HCl buffer solution for annealing treatment, and the conditions of the annealing treatment are as follows: and (3) reducing the temperature to 16-28 ℃ at the speed of 0.5 ℃/min after 5min at 95 ℃, and more preferably reducing the temperature to 25 ℃ to obtain the carrier with the three-fork type self-assembly structure. In the invention, the annealing operation can enable three DNA self-assembly single chains to self-assemble to form a three-fork type self-assembly structure carrier, and particularly, the three DNAs of ASM-DNA-1, ASM-DNA-2 and ASM-DNA-3 are respectively in pairwise complementary pairing by taking the middle as a node and two ends to form a three-fork type self-assembly structure. In the present invention, the Tris-HCl buffer preferably has a pH of 7.4 to 8.0, more preferably 7.8, and a concentration of 30mM, and in the present invention, the Tris-HCl buffer preferably comprises 10mM MgCl2. In the present invention, the volume ratio of the mixture of the DNA self-assembly single strand and the circularized DNA template is preferably 1: (0.8-1.2), more preferably 1:1, in the present invention, the concentration of each of the DNA self-assembled single strand and the circularized DNA template in the buffer is preferably 1.0X 10-4mol/L～1.0×10-7mol/L, more preferably 1.0X 10-6mol/L. In this process, the circular DNA template does not react.
After the three-fork type self-assembly structure carrier is obtained, the three-fork type self-assembly structure carrier is mixed with T4 ligase and T4 ligase buffer solution, and cyclization reaction is carried out at 16 ℃ to obtain the three-fork type self-assembly structure carrier containing the annular template. The sources of the T4 ligase and the buffer solution thereof are not particularly limited in the present invention, and conventional commercially available T4 ligase and the buffer solution thereof can be used. The method of using the T4 ligase in the present invention is not particularly limited either, and a conventional method of using T4 ligase may be employed. In the invention, the 5' ends of the three cyclization DNA templates contain phosphate groups, and the phosphate groups can connect the two ends of a DNA chain, thereby being beneficial to cyclization; and the tail end of the branch of the trifurcate self-assembly structure carrier formed by the DNA self-assembly single strand contains a T7 promoter sequence, the two ends of the circular DNA template are provided with sequences which are complementary and paired with T7, the two sequences interact through base pairing to form a circular template (the circular template respectively consists of respective microRNA complementary sequences and T7 promoter complementary sequences), and the trifurcate self-assembly structure carrier containing the circular template is obtained.
After obtaining the trifurcate self-assembly structure carrier containing the circular template, the invention mixes the trifurcate self-assembly structure carrier containing the circular template with nucleotide triphosphate mixed solution, RNA enzyme inhibitor, T7 polymerase and T7 polymerase buffer solution, and carries out rolling circle transcription reaction for 0.5-3 h, more preferably 1.5h at 37 ℃ to obtain the carrier combined with circular DNA. The sources, the amounts and the methods of using the nucleotide triphosphate mixture, the rnase inhibitor, the T7 polymerase and the buffer thereof are not particularly limited, and conventional commercial products, conventional amounts and conventional methods of use known to those skilled in the art may be used. In the invention, the rolling circle transcription Reaction (RCT) takes a trifurcate self-assembly structure carrier containing an annular template as a template, and amplification of the annular DNA template is realized through reaction at 37 ℃ for 0.5-3 h. Amplification of this circularized DNA template facilitates binding of the targeting aptamer.
After the carrier combined with the circular DNA is obtained, the carrier combined with the circular DNA is mixed with a targeting aptamer to carry out annealing treatment, wherein the annealing treatment conditions are as follows: and (3) reducing the temperature to 16-28 ℃ at the speed of 0.5 ℃/min after 5min at the temperature of 65 ℃, and more preferably reducing the temperature to 25 ℃ to obtain the RNA nano hydrogel for the targeted therapy of the lung cancer. In the invention, the volume ratio of the vector combined with the circular DNA to the targeting aptamer is preferably 1 (90-110), more preferably 1: 100. in the invention, in the annealing process, the targeting aptamer is complementarily combined with a T7 promoter sequence in the amplified circular DNA template to obtain the RNA nano hydrogel for the targeted therapy of the lung cancer.
The invention also provides application of the RNA nano hydrogel in the technical scheme or the RNA nano hydrogel prepared by the preparation method in the technical scheme in preparation of a medicine for targeted inhibition of lung cancer cells.
In the present invention, the lung cancer cells include the a549 cell line. In the present invention, the following method is preferably used for verifying the effect of the RNA hydrogel: the RNA nano hydrogel is acted on an A549 cell line: the RNAHs and the cultured A549 cells are placed in an incubator for incubation under the culture environment condition of 37 ℃ and 5% CO2And (4) carrying out relevant detection after incubation for a certain time.
In the invention, the effective dosage of the RNA nano hydrogel in the medicine is 30-150 mu L.
The RNA nano-hydrogel for targeted therapy of lung cancer, the preparation method and the application thereof according to the present invention are further described in detail with reference to the following embodiments, and the technical solutions of the present invention include, but are not limited to, the following embodiments.
Example 1
Preparation of RNA nano hydrogel:
three DNA single strands (ASM-DNA-1, ASM-DNA-2 and ASM-DNA-3) for synthesizing the Three-fork self-assembly structure carrier and Three cyclization DNA templates of Three-let7a, Three-miR34a and Three-miR 145 for cyclization are mixed together respectively, and Tris-HCl buffer solution is added to the mixture to ensure that the concentration of the substances is 1 multiplied by 10 to be constant to 20 mu L-6mol/L. Annealing the mixed solution (95 ℃, 5min, then reducing the temperature by 0.5 ℃ per minute to 25 ℃), and then preparing the three-fork self-assembly structure carrier;
adding T4 ligase and its buffer solution, and placing them at 16 deg.C for cyclization to obtain the trifurcate self-assembly structure carrier containing circular template.
Then adding nucleotide triphosphate mixed solution (rNTP), an RNase inhibitor, T7 polymerase and buffer solution thereof to carry out RCT reaction for 1.5h at 37 ℃ to obtain a carrier combined with circular DNA;
mixing the raw materials in a ratio of 1: 100, mixing the carrier combined with the circular DNA and the targeting aptamer, and carrying out annealing treatment, wherein the conditions of the annealing treatment are as follows: and (3) reducing the temperature to 25 ℃ at the speed of 0.5 ℃/min after 5min at the temperature of 65 ℃ to obtain the RNA nano hydrogel for the targeted therapy of the lung cancer.
Example 2
The successful synthesis of the RNA nano-hydrogels (RNANHs) prepared in example 1 was characterized and verified by agarose gel electrophoresis, which specifically comprises the following steps:
first, agarose (2% by mass) was dissolved in TAE buffer and heated to boiling in a microwave oven, then gel-stained with ethidium bromide stain until the gel was completely solidified, and then the solidified gel was transferred to an electrophoresis apparatus filled with TAE buffer. Then, products (a three-fork type self-assembly structure carrier, a three-fork type self-assembly structure carrier containing a circular template, and an RNA nano hydrogel for targeted therapy of lung cancer) of each step in the experiment implementation steps are mixed with a gel loading dye containing bromophenol blue, and then the mixture is dripped into gel holes, and the experiment is carried out for 40min under the voltage of 110V.
The invention observes and records gel images under an ultraviolet gel imager. Morphology of RNANHs was characterized using projection electron microscopy (TEM). The electrophoresis characterization picture of the RNA nano hydrogel and the TEM detection result are shown in FIG. 2, wherein FIG. 2-1 is the electrophoresis characterization picture of the RNA nano hydrogel, and FIG. 2-2 is the TEM detection result picture. As can be seen from FIG. 2-1, the present invention has succeeded in synthesizing a product having a relatively large molecular weight; the morphological structure and particle size of the product can be seen from FIG. 2-2.
The results show that after the formation of the three-fork type self-assembly structure carrier, the T4 ligase acts, and the three-fork type self-assembly structure carrier containing the circular template is successfully synthesized. And the final RNA nano hydrogel, namely the product RNANHs has higher molecular weight and is successfully synthesized. According to TEM analysis, the average particle size of RNANHs is about 150 nm.
Example 3
Particle size of RNANHs was measured with a particle sizer:
the comparison result of the particle size of the RNA nano hydrogel before and after the addition of the cholesterol-modified targeting aptamer is shown in FIG. 3, and the detection is carried out by a particle size analyzer, wherein FIG. 3-1 shows that a product directly obtained after RCT transcription, namely a carrier combined with circular DNA, is a particle with the average diameter of about 1 μm, but after the FAM-S6apt-CHOL targeting aptamer is added, the particle size of the obtained RNAHs is reduced to about 150nm as shown in FIG. 3-2. It is thus clear that cholesterol modified on the fragment FAM-S6apt-CHOL, a targeting aptamer, can make the structure of RNANHs more compact and thus reduce the particle size due to its hydrophobic nature.
Example 4
Verifying the target recognition effect of the nano hydrogel (RNANHs) on cells under the action of aptamers, and performing detection analysis through CLSM and a flow cytometer, wherein the specific process comprises the following steps:
pre-culturing A549 cells, HeLa cells and L02 cells in a glass button cell culture dish (phi is 15mm) for 12h for standby; and washing the cells twice by using PBS buffer solution, then culturing the cells for 2 hours in a 37 ℃ culture box by using a culture medium containing RNAHs, washing the cells for not less than three times by using the PBS buffer solution after the incubation time is finished, and detecting the cells by using a laser scanning confocal microscope (CLSM). For the flow cytometry detection, after the cell sample to be detected after the incubation is finished is digested with pancreatin, the cell sample is resuspended in a centrifuge tube of 1.5mL by PBS for detection.
The CLSM detection graph of the RNA nano hydrogel on different cell target recognition effects is shown in FIG. 4, wherein FIGS. 4-1 to 4-3 are CLSM fluorescence imaging graphs of A549 cells, Hela cells and L02 cells respectively, and FIGS. 4-4 to 4-6 are corresponding dark-field CLSM graphs (scale is 50 μm) respectively. As shown in the fluorescence detection chart of FIG. 4, it can be seen from CLSM imaging detection that RNAHs have a good specific recognition effect on the A549 cells of the target cells, and can enter the cells to detect fluorescence, while the comparative HeLa and L02 cells have no obvious fluorescence signals.
The flow analysis of the RNA nano-hydrogel for different cell target recognition is shown in FIG. 5 (FIGS. 5-1 to 5-3 are flow analysis of RNA nano-hydrogel after it acts on A549 cell, Hela cell and L02 cell, respectively). Analysis shows that the RNAHs can realize right shift of peak only after the RNAHs reacts with the A549 cells, and a relatively obvious fluorescence signal is detected, while the HeLa and L02 cells have no change of the fluorescence signal after being cultured by the RNAHs. The results effectively prove the specific target recognition effect of RNANHs on A549 cells.
Example 5
Cytotoxicity experiments were performed using the CCK-8 kit:
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)]X 100%, and is shown in FIG. 6 after being represented by a graph.
The results of the cytotoxicity tests of the RNA nano hydrogel containing a single ring-forming DNA template, the RNA nano hydrogel containing three ring-forming DNA templates and the hydrogel containing no microRNA on A549 cells respectively are shown in FIG. 6. The results show that under the same experimental conditions, the cell survival rate after the action of RNANHs is obviously lower than that after the action of single RNA hydrogel only containing microRNA, but the cell survival rate after the action of the single RNA hydrogel containing microRNA is lower than that of a control group (RNA nano hydrogel without microRNA). The RNA nano hydrogel containing the microRNA has a targeted inhibition effect on the lung cancer cells, and the RNA nano hydrogel containing the three microRNAs has the relatively best treatment effect on the targeted lung cancer cells.
The RNA nano hydrogel containing microRNA for gene regulation can be effectively transferred into target cells to play a role under the action of an aptamer, so that the aim of inhibiting the growth of tumor cells and even killing the tumor cells is fulfilled. The RNA nano hydrogel nanotechnology system provided by the invention provides a novel comprehensive treatment method, and effectively transfers microRNA into targeted cells to play a therapeutic role.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
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Claims (10)

1. An RNA nano hydrogel for targeted therapy of lung cancer, which is characterized by comprising a DNA self-assembly single strand, a ring-forming DNA template and a targeted aptamer;
the DNA self-assembly single strand comprises ASM-DNA-1 with a nucleotide sequence shown as SEQ ID NO.1, ASM-DNA-2 with a nucleotide sequence shown as SEQ ID NO.2 and ASM-DNA-3 with a nucleotide sequence shown as SEQ ID NO.3, the three DNA single strands respectively take the middle of the sequences as a node, and two ends of the three DNA single strands are in pairwise complementary pairing to form a three-fork self-assembly structure;
the loop-forming DNA template comprises a nucleotide sequence with phosphorylated 5 ' end shown as Three-let7a shown as SEQ ID NO.4, a nucleotide sequence with phosphorylated 5 ' end shown as Three-miR34a shown as SEQ ID NO.5 and a nucleotide sequence with phosphorylated 5 ' end shown as Three-miR 145 shown as SEQ ID NO.6, and is complementarily combined with a T7 promoter sequence at the branch end of the Three-fork self-assembly structure through T7 promoter complementary sequences at two ends of the sequences;
the targeting aptamer is a substance of which the 5 'end of the sequence of SEQ ID NO.7 is modified with a fluorescent group and the 3' end is modified with cholesterol; the targeting aptamer binds to the circularized DNA template through the T7 promoter complement.
2. The RNA nano-hydrogel according to claim 1, wherein the RNA nano-hydrogel has a particle size of 80-200 nm.
3. The RNA nanohydrogel of claim 1, wherein the fluorophore comprises FAM, FITC or Cy 3.
4. The method for preparing the RNA nano hydrogel of any one of claims 1 to 3, which comprises the following steps:
1) mixing a DNA self-assembly single strand and a ring-forming DNA template in a Tris-HCl buffer solution for annealing treatment, wherein the annealing treatment conditions are as follows: reducing the temperature to 16-28 ℃ at the speed of 0.5 ℃/min after 5min at 95 ℃ to obtain a three-fork self-assembly structure carrier;
2) mixing the trifurcate self-assembly structure carrier obtained in the step 1) with T4 ligase and T4 ligase buffer solution, and performing cyclization reaction at 16 ℃ to obtain a trifurcate self-assembly structure carrier containing an annular template;
3) mixing the trifurcate self-assembly structure carrier containing the circular template obtained in the step 2) with a nucleotide triphosphate mixed solution, an RNase inhibitor, T7 polymerase and T7 polymerase buffer solution, and carrying out rolling circle transcription reaction at 37 ℃ for 0.5-3 h to obtain a carrier combined with circular DNA;
4) mixing the circular DNA-combined carrier obtained in the step 3) with a targeting aptamer, and carrying out annealing treatment under the conditions of: and (3) reducing the temperature to 16-28 ℃ at the speed of 0.5 ℃/min after 5min at 65 ℃ to obtain the RNA nano hydrogel for the targeted therapy of the lung cancer.
5. The method according to claim 4, wherein the Tris-HCl buffer of step 1) has a pH of 7.4 to 8.0.
6. The method according to claim 4, wherein the mixing volume ratio of the DNA self-assembly single strand to the circularized DNA template is 1: 0.8-1.2.
7. The method according to claim 4, wherein the mixing volume ratio of the circular DNA-binding vector to the targeting aptamer in step 4) is 1: (90-110).
8. Use of the RNA nano-hydrogel according to any one of claims 1 to 3 or the RNA nano-hydrogel obtained by the preparation method according to any one of claims 4 to 7 in preparation of a drug for targeted inhibition of lung cancer cells.
9. The use of claim 8, wherein the lung cancer cells comprise the a549 cell line.
10. The use of claim 8, wherein the effective amount of the RNA nano-hydrogel in the medicament is 30-150 μ L.
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