CN115595325A - SaRNA of target miR-181a promoter and application thereof in preparation of medicine for treating CML - Google Patents

Abstract

The invention discloses a saRNA of a target miR-181a promoter and application thereof in preparing a medicament for treating chronic granulocytic leukemia, wherein the saRNA is saRNA1, saRNA2 and saRNA3; the saRNA can promote the expression of miR-181a in CML sensitive and CML drug-resistant cells, and simultaneously the protein expression level of target protein PPFIA1 of miR-181a is reduced compared with that of an NC group. The sarRNA can overcome the CML drug resistance independent of BCR-ABL in cell and mouse models, and the inhibition effect is better than miR-181a imic.

Description

SaRNA of target miR-181a promoter and application thereof in preparation of medicine for treating CML

Technical Field

The invention belongs to the field of biological medicines, and particularly relates to a miR-181a promoter-targeted sarRNA and application thereof in preparation of medicines for treating Chronic Myelocytic Leukemia (CML).

Background

Chronic myeloid leukemia is a hematological malignancy, and BCR-ABL1 fusion protein is the core role in pathogenesis of chronic myeloid leukemia, leading to clonal expansion of hematopoietic cells.

In the treatment of Chronic Myeloid Leukemia (CML), patients are prone to imatinib resistance, which presents a challenge to clinical treatment.

The drug resistance of CML patients can be divided into two cases, based on whether ABL1 is mutated or not, depending on BCR-ABL drug resistance and independent of BCR-ABL drug resistance. Reasons for BCR-ABL dependent resistance may include: (1) ABL1 in BCR-ABL1 is mutated, most commonly T315I; (2) BCR-ABL1 overexpression.

After the second generation and third generation Tyrosine Kinase Inhibitor (TKI) drugs are marketed, the clinical treatment of drug-resistant CML dependent on BCR-ABL1 has achieved good results. However, in the drug-resistant CML treatment independent of BCR-ABL1, the clinical challenge of the drug-resistant symptom is larger because the pathogenesis is complex and diversified, and the clinical treatment does not have an effective and uniform treatment scheme.

Disclosure of Invention

The invention aims to provide a miR-181a promoter-targeted sarRNA and application thereof in preparation of a medicine for treating chronic granulocytic leukemia.

The purpose of the invention is realized by the following technical scheme:

the sarnas targeting the miR-181a promoter are saRNA1, saRNA2 and saRNA3, and the sequences are shown as follows:

saRNA1：

sense:5’-GGAAUAUGAUGAUAAUUUA dTdT-3’

antisense:5’-UAAAUUAUCAUCAUAUUCC dTdT-3’；

saRNA2：

sense:5’-GGGCUGUAAUUUAGUUCAA dTdT-3’

antisense:5’-UUGAACUAAAUUACAGCCC dTdT-3’；

saRNA3：

sense:5’-GGUAGUUUACAGCCUCUAA dTdT-3’

antisense:5’-UUAGAGGCUGUAAACUACC dTdT-3’。

the saRNA targeting the miR-181a promoter is applied to the preparation of a medicament for treating chronic granulocytic leukemia;

the chronic granulocytic leukemia comprises imatinib-sensitive and imatinib-resistant chronic granulocytic leukemia;

the chronic granulocytic leukemia sensitive to imatinib particularly refers to K562 and KCL22 cell strains;

the imatinib-resistant chronic granulocytic leukemia comprises BCR-ABL 1-dependent and independent drug-resistant chronic granulocytic leukemia;

the BCR-ABL1 independent drug-resistant chronic granulocytic leukemia refers to imatinib-resistant cell lines without any one of the following mutations: T315I, E255K, E255V, G E, Q H, Y H;

the imatinib resistant cell strain particularly refers to K562-IMR and KCL22-IMR;

the medicine also contains other active ingredients and auxiliary materials (carriers);

the auxiliary materials (carriers) are preferably sustained-release agents, excipients, fillers, adhesives, wetting agents, disintegrating agents, absorption promoters, adsorption carriers, surfactants or lubricants and the like;

the medicament can be prepared into aerosol, tablets, capsules, dripping pills, powder, solutions, suspensions, emulsions, granules, lipidic agents, transdermal agents, buccal agents, suppositories or freeze-dried powder injections and the like.

When the sarRNA of the miR-181a promoter is targeted to act, under the condition of the same concentration, compared with miR-181a mimic, the action duration of the sarRNA is longer, and the growth of CML cells can be inhibited more effectively. The effect of RNA-activated single transfection of saRNA is prolonged. RNA activation can activate endogenous genes. Among tumors and malignant diseases, saRNA is expected to develop a new target drug. The saRNA designed according to the rules can enhance the expression of tumor suppressor genes and restore the natural functions of the genes. Small nucleic acid drugs are generally two-dimensional structures that do not themselves encode proteins, and are more versatile and diverse than protein target drugs. The saRNA can accelerate the development of applicable drugs, and provides possible treatment channels and cure hopes for more malignant diseases or rare diseases.

Compared with the prior art, the invention has the following advantages and effects:

the saRNAs targeting the miR-181a promoter can improve the expression level of endogenous original miR-181a (pri-miR-181 a), and pri-miR-181a has a stronger inhibitory effect than miR-181a micic.

Drawings

FIG. 1 shows the proliferation rate of K562 cells.

FIG. 2 is the expression level of miR-181a in K562 cells after 1-3 72h transfection of sarRNA.

FIG. 3 is the protein expression level of miR-181a target gene PPFIA1 in K562 cells 3h after transfection of sarRNA.

FIG. 4 shows the proliferation inhibition rate of SARNA3 and miR-181a on K562 cells.

FIG. 5 is clonogenic and statistical analysis of K562 cells.

FIG. 6 is the SNP sequencing results of K562-IMR and KCL22-IMR; wherein, A: K562-IMR; b: KCL22-IMR.

FIG. 7 shows the inhibition rate of proliferation of CML-resistant cells by sarRNA-3; wherein, A: K562-IMR; b: KCL22-IMR.

FIG. 8 is the expression level of miR-181a and the protein expression level of PPFIA1 in CML-resistant cells after transfection of sarRNA 3.

FIG. 9 is a clone formation and statistics of CML resistant cells.

FIG. 10 shows that K562-IMR/luciferas cells stably expressed luciferase wherein A: the correspondence between the luminous intensity and the number of cells; b: the imaging brightness corresponds to the number of cells.

FIG. 11 shows the construction of a drug-independent resistant CML mouse model from human K562-IMR/luciferas cell-transplanted B-DNG mice; wherein, A: fluorescence intensity display of B-NDG mice; b: the survival time of B-NDG mice was compared.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

Examples

SaRNA for designing and activating expression of target miR-181a and effect thereof in treatment of sensitive and drug-resistant CML

1.1 designing sarRNA for activating miR-181a expression

The promoter region of the target gene was retrieved and identified, and MIR181A1HG was located on chromosome 1 and was approximately 129kb in length. Designing saRNA (small activating RNA) aiming at the promoter thereof:

saRNA1：

sense:5’-GGAAUAUGAUGAUAAUUUA dTdT-3’

antisense:5’-UAAAUUAUCAUCAUAUUCC dTdT-3’；

saRNA2：

sense:5’-GGGCUGUAAUUUAGUUCAA dTdT-3’

antisense:5’-UUGAACUAAAUUACAGCCC dTdT-3’；

saRNA3：

sense:5’-GGUAGUUUACAGCCUCUAA dTdT-3’

antisense:5’-UUAGAGGCUGUAAACUACC dTdT-3’。

1.2 screening of RNA1-3 for inhibition of K562 cell viability

The K562 cells are from Shanghai cell bank of Chinese academy of sciences;

and (3) transfecting K562 cells with 200nM sarnA1-3 or miR-181a mimics, detecting the cell activity by a CCK-8 method 72 hours after transfection, and evaluating the inhibition rate.

The specific procedures for transfection were:

1) And taking the CML cells with good growth condition, and counting by using a cell counting plate. Adding RPMI-1640 medium without serum and antibiotic into the centrifugal tube after calculation, uniformly mixing and diluting the mixture at 3X 10 ⁵ The number of cells/well is inoculated on a 6-well plate;

2) Setting groups of BK (blank control group), NC (negative control group), miR-181a imic group and saRNA1-3 group, wherein each group is provided with 3 multiple wells, and the transfection concentration is 100nM;

NC is random RNA duplex, sequence:

sense:5’-uucuccgaacgugucacgutt-3’

antisense:5’-acgugacacguucggagaatt-3’；

the sequence of miR-181a micic is as follows: 5'-aacauucaacgcugucggugagu-3';

3) Taking a sterile EP tube, and placing at 5 μ L/wellAmount of Lipofectamine ^TM 2000, add Opti-MEM at 250. Mu.L/well for dilution, and mix well slowly. Sucking 20 μ M nucleic acids of each group by 10 μ L/well on EP tube according to group, adding Opti-MEM for dilution according to 250 μ L/well, and standing at room temperature for 5min in ultra-clean bench;

4) According to dilution Lipofectamine ^TM 2000 and diluted nucleic acid volume 1:1, sucking, gently and fully mixing uniformly, and incubating at room temperature for 20min;

5) After incubation was complete, 500. Mu.L of Lipofectamine per well was aspirated ^TM 2000-RNA complexes were carefully and slowly added to the plated cells;

6) The 6-well plate was placed in a cell incubator at 37 ℃ and 5% by volume and allowed to stand. The transfection was completed after 6h of standing.

The result is shown in figure 1, all the sarnas 1 to 3 can inhibit the proliferation of K562 cells, but the inhibition effect of the sarnas 3 on the K562 cells is stronger than that of miR-181a mimic. Compared with the BK group, the average inhibition rate of the NC group is 16.40%, the average inhibition rate of the miR-181a group is 26.56%, the average inhibition rate of RNA-1 is 25.60%, the average inhibition rate of RNA-2 is 25.96%, and the average inhibition rate of sarRNA-3 is 33.43% (the improvement is 17.03%). As shown in figure 1, the inhibition effect of the sarRNA-3 on K562 cells is stronger than that of miR-181a on the K562 cells.

1.3 detecting whether expression of miR-181a is activated

After transfection with saRNA1-3, the expression level of miR-181a in K562 cells was detected.

As shown in FIG. 2, only sarRNA-3 promoted the expression of miR-181a, as compared with the NC group. Neither saRNA-1 nor saRNA-2 can activate miR-181a and increase its expression.

Transfected saRNA-3 was selected and compared to NC groups for WB experiments in K562 cells. As shown in FIG. 3, after transfection of sarRNA-3, the protein expression level of the target protein PPFIA1 of miR-181a is reduced compared with that of the NC group.

1.4 comparison of the duration of action of miR-181a and sarRNA 3 on K562 cells

After K562 cells were transfected with sarRNA 3 or miR-181a, the inhibition effect on cell proliferation at 72h, 96h, 120h and 144h was examined with CCK-8.

The results are shown in FIG. 4, and the inhibition effect of sarRNA 3 on the proliferation of K562 cells is stronger than that of miR-181a. At 72 hours, saRNA3 is increased by 14.71 percent of inhibition rate compared with miR-181 a; at 96 hours, the inhibition rate of saRNA3 is increased by 14.81 percent compared with miR-181 a; at 120h and 144h, miR-181a has no statistical significance compared with the NC group, but sarRNA 3 still has statistical significance compared with the NC group. The experiment shows that the inhibition effect of the saRNA-3 on the K562 is longer.

1.5 Effect of saRNA3 on clonogenic Capacity of K562 cells

The proliferation capacity of K562 cells can be assessed by soft agar colony formation experiments.

Experimental setup groups were respectively: blank control group (Blank), 100nM NC (negative control), 100nM sarnA3 (i.e., sarRNA-3), 3 duplicate wells per group were set.

1) After counting, the cells are diluted by serum-free and double-resistant-free RPMI-1640 culture medium and inoculated in a 96-well plate at the rate of 5000 cells/50 mu L per well;

2) Aspirate Lipofectamine at 0.25. Mu.L/well ^TM 2000, add the serum-reduced culture medium Opti-MEM at 25. Mu.L/well, and mix well slowly. Pipetting 20 μ M nucleic acid of each group at 0.5 μ L/well according to group, diluting with 25 μ L/well in Opti-MEM, and incubating at room temperature for 5min;

3) According to dilution Lipofectamine ^TM 2000 and diluted nucleic acid volume 1:1, sucking, gently and fully mixing uniformly, and incubating at room temperature for 20min; aspirate 50. Mu.L Lipofectamine per well ^TM The 2000-RNA compound is slowly dripped into the cell suspension;

4) The 96-well plate was placed in a cell incubator at 37 ℃ and 5% by volume, and allowed to stand. Finishing transfection after placing 6 h;

5) A sterile centrifuge tube was poured into 25mL of 1.2% soft agarose gel solution (incubation), then 25mL of 20% FBS 1640 medium was added (incubation), and the mixture was mixed well. Adding 1mL of the mixture (containing 0.6% soft agarose gel solution and 10% FBS 1640 medium) into each well of a 6-well plate, and cooling and solidifying to obtain a lower layer gel;

6) A sterile centrifuge tube is taken, 25mL of 0.7% soft agarose gel solution is poured (heat preservation), 25mL of 20% FBS 1640 culture medium is added (heat preservation), and the mixture is fully mixed. Cell suspensions were collected 6h after transfection with small nucleic acids. Each well of cells was mixed with 1mL of supernatant gel mixture (containing 0.4% soft agarose gel solution and 10% FBS 1640 medium), and the mixture was added dropwise to the lower gel and shaken well. Cooling and solidifying to obtain cell-containing upper layer gel, and culturing the gel in a carbon dioxide incubator;

7) When the appearance of clones was observed, the PBS solution was slowly flowed through the gel plane 3 times and the PBS solution was aspirated off. Adding 500 mu L of 4% paraformaldehyde solution into each hole, fixing for 15-30min, and washing the glue plane by PBS for 3 times;

8) Adding 1mL0.005% crystal violet dye solution into each hole, and standing until the colony is dyed;

9) Sucking off 0.005% of crystal violet dye solution, adding PBS (phosphate buffer solution) for repeated washing until the dye solution is washed away;

10 Photographing and storing, and counting the number of clones in each group.

As shown in FIG. 5, the clone formation in the group of BK was comparable to that in the NC group, whereas that in the group of Sara-3 transfected K562 cells was less than that in the NC group. This experiment demonstrates that saRNA-3 inhibits colony formation in K562 cells in a soft agar experiment.

1.6 inhibition of transfection of saRNA-3 on CML drug-resistant cell viability

The K562 cells are from Shanghai cell bank of Chinese academy of sciences; KCL22 cells were donated by professor Muschen at the children hospital los angeles;

and establishing K562-IMR and KCL22-IMR CML drug-resistant cell strains in a drug concentration increasing mode.

And carrying out SNP sequencing on K562-IMR and KCL22-IMR. 6 common mutation sites in BCR-ABL-dependent drug resistance are selected, the base sequence of the sites with frequent mutation in the two drug-resistant cell strains is detected, and no mutation is found (as shown in FIG. 6). K562-IMR and KCL22-IMR are proved to be imatinib-resistant cell strains independent of BCR-ABL 1.

To examine the inhibitory effect of saRNA-3 in CML-resistant cells, the inhibitory effect of transfected saRNA-3 on K562-IMR and KCL22-IMR cell proliferation was compared to NC and measured with CCK 8.

The result is shown in figure 7, the saRNA-3 can inhibit the proliferation of K562-IMR and KCL22-IMR cells, and the inhibition effect of the saRNA-3 on the K562-IMR and KCL22-IMR cells is stronger than that of miR-181a mimic. The inhibition rate of the K562-IMR NC group is 17.42%, compared with the NC group, the inhibition rate of the miR-181a group is 9.00%, and the inhibition rate of the sarRNA-3 group is 28.47%. The inhibition rate of NC group in KCL22-IMR cells is 14.98%, compared with NC group, the inhibition rate of miR-181a group is 11.13%, and the inhibition rate of sarRNA-3 is 22.56%.

The inhibition effect of the sarRNA-3 on K562-IMR and KCL22-IMR cells is stronger than that of miR-181a.

1.7 detecting whether miR-181a is activated by sarRNA-3

After transfection with saRNA-3, expression levels of miR-181a in K562-IMR and KCL22-IMR cells were measured.

Firstly, whether the expression of miR-181a is increased is detected, and as shown in FIG. 8, sarRNA-3 can increase the expression of miR-181a.

Transfected saRNA-3 was selected and compared to NC groups and Western Blot experiments were performed in K562-IMR and KCL22-IMR cells. After the saRNA-3 is transfected, the protein expression level of the target protein PPFIA1 of the miR-181a is reduced compared with that of an NC group.

The fact that the designed saRNA-3 targeting the miR-181a promoter can activate to generate miR-181a is shown by detecting the expression of miR-181a and the expression of PPFIA 1.

1.8 detection of clonogenic Capacity of cells after transfection of saRNA-3

The proliferation capacity of K562-IMR cells and KCL22-IMR cells can be examined by soft agar colony formation experiments. 5000 cells per well were repeatedly seeded in 6-well plates, and after colony formation, crystal violet staining was performed and counted. As shown in FIG. 9, the clones formed after transfection of K562-IMR cells and KCL22-IMR cells with sarRNA-3 were less than those in the NC group, whereas the clones formed in the BK group were comparable to those in the NC group. This experiment demonstrates that saRNA-3 inhibits colony formation of K562-IMR cells and KCL22-IMR cells in a soft agar colony formation experiment.

1.9 Sara-3 treatment inhibits proliferation of K562-IMR/luciferas cells in B-NDG mice

The luciferase reporter gene vector (Beijing Biocytogen) is stably transferred to K562-IMR cells, and the K562-IMR cells are screened by puromycin and maintained and cultured for 1 week to obtain the K562-IMR/luciferase cells. K562-IMR/luciferase cells were diluted in complete medium (RPMI-1640 medium supplemented with 10% fetal bovine serum, 1% double antibody), planted in 96 Kong Quanbai opaque plates in different numbers of K562-IMR/luciferase cells, added with 200 Xluciferase substrate and incubated for 30min. After incubation, detection was performed with a multifunctional microplate reader, and the experimental results are shown in fig. 10A.

The plate testing mode is adjusted in a small animal living body luminescence imaging system, different K562-IMR/luciferase cell numbers are planted in a 96 Kong Quanbai light-tight plate, and the experimental results obtained by detection are shown in FIG. 10B. Experimental results show that the more the number of the K562-IMR/luciferase cells, the stronger the fluorescence intensity detected by the instrument, and that the K562-IMR/luciferase cells can act on a luciferase substrate and emit fluorescence and can be used for tracing the K562-IMR/luciferase cells in subsequent in vivo of the B-NDG mouse.

B-NDG (NOD. CB17-Prkdc) at 4 weeks of age ^scid IL2rg ^tm1 /Bcgen) mice were purchased from beijing baiosu map, B-NDG mice were genetically background on NOD-scid, where the Il2rg gene was knocked out, and mice of this species lacked mature T, B and NK cells.

Two weeks after quarantine of the purchased mice, each B-NDG mouse was injected by tail vein with 1X 10 ⁶ K562-IMR/luciferase cells; on the day after injection (day 0), in vivo imaging assays were performed on each B-NDG mouse, confirming that K562-IMR/luciferas cells were injected into the B-NDG mice via tail vein injection. After B-NDG is attacked, hair is withered and lusterless, the body is thin, the appetite is poor, and the phenomenon of bow and back appears.

From the 10 th day after injection, mice are treated in groups (3 mice in each group), the mice are treated with medicaments every other day, living body imaging detection is carried out after treatment, and the proliferation and transfer conditions of K562-IMR/luciferase cells in the mice are observed; the treatment groups were as follows:

(1) NC group: each B-NDG mouse was given 200. Mu.L of 10nmol NC solution.

(2) saRNA-3 group: each B-NDG mouse was given 200. Mu.L of a 10nmol sarRNA-3 solution.

Recording the death time of the B-NDG mice, weighing the body weight of each group of B-NDG mice, recording the change, and making survival curves of the B-NDG mice treated by the drug in different groups.

As a result, as shown in FIG. 11, in comparison with the NC group, the bioluminescence intensity representing the degree of morbidity was reduced in the sarRNA-3 group, and the survival time of the mice was longer (the survival time of the B-NDG mice could be prolonged by about 5 days). Experimental results show that the development of CML drug resistance independent of BCR-ABL1 can be overcome to a certain extent by the sarRNA-3 proved by B-NDG mouse in vivo experiments.

The results show that the saRNA-3 can promote the expression of miR-181a in CML sensitive and CML drug-resistant cells, and the protein expression level of the target protein PPFIA1 of miR-181a is reduced compared with that of an NC group. The sarRNA-3 can overcome the CML drug resistance independent of BCR-ABL in cell and mouse models, and the inhibition effect is better than miR-181a imic.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims (9)

1. The saRNA targeting the miR-181a promoter is characterized in that: the sequences of the saRNA1, the saRNA2 and the saRNA3 are shown as follows:

saRNA1：

sense:5’-GGAAUAUGAUGAUAAUUUA dTdT-3’

antisense:5’-UAAAUUAUCAUCAUAUUCC dTdT-3’；

saRNA2：

sense:5’-GGGCUGUAAUUUAGUUCAA dTdT-3’

antisense:5’-UUGAACUAAAUUACAGCCC dTdT-3’；

saRNA3：

sense:5’-GGUAGUUUACAGCCUCUAA dTdT-3’

antisense:5’-UUAGAGGCUGUAAACUACC dTdT-3’。

2. the application of the saRNA targeting the miR-181a promoter of claim 1 in preparing a medicament for treating chronic myelogenous leukemia.

3. Use according to claim 2, characterized in that: the chronic granulocytic leukemia comprises imatinib-sensitive and imatinib-resistant chronic granulocytic leukemia.

4. Use according to claim 3, characterized in that: the chronic granulocytic leukemia sensitive to imatinib refers to K562 and KCL22 cell strains.

5. Use according to claim 3, characterized in that: the imatinib-resistant chronic granulocytic leukemia comprises BCR-ABL 1-dependent and independent drug-resistant chronic granulocytic leukemia;

the BCR-ABL1 independent drug-resistant chronic granulocytic leukemia refers to imatinib-resistant cell lines without any one of the following mutations: T315I, E255K, E255V, G E, Q62252H, Y H.

6. Use according to claim 5, characterized in that: the BCR-ABL1 independent drug-resistant chronic granulocytic leukemia refers to K562-IMR and KCL22-IMR cell strains.

7. Use according to claim 2, characterized in that: the medicine also contains other active ingredients and auxiliary materials.

8. Use according to claim 7, characterized in that: the auxiliary materials are sustained release agents, excipients, fillers, adhesives, wetting agents, disintegrants, absorption enhancers, adsorption carriers, surfactants or lubricants.

9. Use according to claim 7, characterized in that: the medicament can be prepared into aerosol, tablets, capsules, dripping pills, powder, solutions, suspensions, emulsions, granules, lipidic agents, transdermal agents, buccal agents, suppositories or freeze-dried powder injections.

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