Application of miRNA-98-3p in preparation of anti-angiogenesis drugs

Abstract

The invention relates to the technical field of biomedicine, and discloses an application of miRNA-98-3p in preparation of an anti-angiogenesis drug, the miRNA-98-3p and MALAT1 are ceRNA for each other in ovarian cancer, and can inhibit the expression of VEGFA so as to inhibit the growth of HUVEC cells, reduce the migration ability of the HUVEC, effectively inhibit the angiogenesis and node formation ability of the HUVEC, and improve the anti-angiogenesis ability of the HUVEC. Tumor angiogenesis is inhibited, and blood perfusion amount is reduced to inhibit in-vivo tumor progression. The invention provides a novel anti-angiogenesis miRNA (micro Ribonucleic Acid) medicine which has a great application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to the application of miRNA-98-3p in the preparation of anti-angiogenic drugs. Background Art

[0002] The statements in this section only provide background information related to the disclosure of the present application and may not constitute prior art.

[0003] Angiogenesis is crucial for maintaining normal physiological processes, such as embryonic development and wound healing, and also plays an important role in the pathogenesis of various diseases, including diabetic retinopathy, rheumatoid arthritis, and cancer.

[0004] Anti-angiogenic drugs play a key role in the treatment of various solid tumors. They act on the tumor microenvironment to degrade existing tumor blood vessels and simultaneously inhibit the formation of new tumor blood vessels, thereby inhibiting tumor growth and metastasis. For example, anlotinib, as an anti-angiogenic drug, has shown good clinical efficacy in the treatment of non-small cell lung cancer (NSCLC). Anti-angiogenic drugs can also be used in combination with other systemic therapeutic drugs (including chemotherapy, small molecule targeted therapy, and immunotherapy) to exert better anti-tumor effects, delay drug resistance, and have controllable adverse reactions.

[0005] Anti-angiogenic drugs are mainly divided into three categories: macromolecular monoclonal antibody drugs, small molecule inhibitors, and pan-target recombinant human endostatin, etc. miRNA (MicroRNA) is a class of single-stranded non-coding RNA molecules that are 21-23 nucleotides long. They regulate various stages of angiogenesis by targeting multiple genes in signaling pathways. Therefore, miRNA has important potential in the development of anti-angiogenic drugs. Their regulatory effects in endothelial cells and their roles in the angiogenesis signaling pathway have been widely studied and may target regulatory molecules such as cytokines, matrix metalloproteinases, and growth factors, including VEGF, PDGF, FGF, EGF, HIF-1, MMP9, MMP2, etc.

[0006] miRNA drugs have better advantages than chemical drugs in anti-angiogenesis, including multi-target effects, simple production and preparation processes, long-lasting drug effects, the ability to achieve breakthroughs for special protein targets that are difficult to develop drugs for, and the great potential to develop therapeutic drugs for "non-targetable" and "non-druggable" diseases. Therefore, developing new types of miRNA drugs is of great significance for disease treatment. Currently, the miRNAs found to be effective in anti-angiogenesis include miR-186, miR-29b, miR-9, and miR-378a, each exerting anti-angiogenic effects through different mechanisms. The types are few, and there is still an urgent need to discover more effective miRNA drugs.

[0007] In view of this, the present application is proposed. Summary of the Invention

[0008] The object of the present invention is to provide an application of miRNA-98-3p in the preparation of an anti-angiogenic drug, aiming at the problem of the small variety of miRNA drugs for anti-angiogenic drugs in the current field, to provide a new anti-angiogenic miRNA drug, and to provide a new optional drug for tumor treatment.

[0009] The technical solution of the present invention is as follows:

[0010] On the one hand, the present application provides an application of miRNA-98-3p in the preparation of an anti-angiogenic drug, and the sequence of the miRNA-98-3p is: CUAUACAACUUACUACUUUCCC.

[0011] According to a preferred embodiment, the miRNA-98-3p is a VEGFA inhibitor when used for anti-angiogenesis.

[0012] On the other hand, the present invention provides an application of miRNA-98-3p mimics in the preparation of an anti-angiogenic drug.

[0013] On the other hand, the present invention provides an application of a miRNA-98-3p expression promoter in the preparation of an anti-angiogenic drug.

[0014] On the other hand, the present invention provides an anti-angiogenic drug composition, which includes miRNA-98-3p, miRNA-98-3p mimics or a miRNA-98-3p expression promoter.

[0015] According to a preferred embodiment, the single-use dose of miRNA-98-3p and miRNA-98-3p mimics in the anti-angiogenic drug composition is: 150 nM.

[0016] According to a preferred embodiment, the drug also contains the medicinal excipients required for the drug.

[0017] According to a preferred embodiment, the excipients are sustained-release agents, fillers, binders, wetting agents, disintegrants, absorption promoters, adsorption carriers, surfactants or lubricants.

[0018] According to a preferred embodiment, the dosage form of the drug is an aerosol, tablet, capsule, pill, powder, solution, suspension, emulsion, granule, lipid agent, buccal tablet, suppository or injection.

[0019] According to a preferred embodiment, the miRNA-98-3p is natural, synthetic, or a DNA fragment and expression vector that express the same miRNA-98-3p sequence.

[0020] The beneficial effects of the present invention compared with the existing technology are as follows:

[0021] 1. On the one hand, the present invention provides a new anti-angiogenic miRNA drug, and it is creatively discovered that miRNA-98-3p can inhibit tumor angiogenesis and reduce the blood perfusion volume of tumors in a mouse model of ovarian cancer, thereby inhibiting tumor development.

[0022] 2. On the one hand, the present invention discovers that miRNA-98-3p can regulate the expression of VEGFA and thus affect tumor angiogenesis; thereby, the application of miRNA-98-3p in anti-angiogenic drugs and tumor treatment is discovered. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Among them, A is the result graph of RNA-seq after knocking down MALAT1 in ovarian cancer cell line HEY-T30 in Example 1, and B is the schematic diagram of the predicted binding sites between these altered miRNAs and MALAT1 on the TargetScan website;

[0024] Figure 2 is the dual-luciferase reporter assay for the binding of miRNA and MALAT1, and the results show that the binding of miR-3611, miR-98-3p, miR-4667-3p, and miR-6886-3p to MALAT1 can all weaken the fluorescence intensity;

[0025] Figure 3 is the real-time RT-qPCR result after knocking down MALAT1 in HEY-T30 cells;

[0026] Figure 4 is the schematic diagram of the result of RNA-seq to detect target genes after knocking down MALAT1 in HEY-T30 cells;

[0027] Figure 5 is the Western blot result after knocking down MALAT1 in HEY-T30 and SKOV3 cells in Example 1;

[0028] Figure 6 is the survival analysis data graph of predicting MALAT1 and VEGFA in ovarian cancer using the Kaplan-Meier Plotter website in Example 1;

[0029] Figure 7Schematic diagram for predicting the binding site of VEGFA and miRNA-98-3p;

[0030] Figure 8 Schematic diagram for the relative luciferase activity in HEK-293T cells co-transfected with miRNA-98-3p mimics and wild-type VEGFA 3'UTR;

[0031] Figure 9 Effect of miRNA-98-3p mimics and inhibitors on the mRNA expression level of VEGFA in ovarian cancer cells HEY-T30 and SKOV3;

[0032] Figure 10 Schematic diagram for the growth effect of knocking down MALAT1 or overexpressing miRNA-98-3p on HUVEC;

[0033] Figure 11 Result diagram of the scratch experiment in Example 2;

[0034] Figure 12 Data diagram of the scratch experiment in Example 2;

[0035] Figure 13 Result diagram of the effect of knocking down MALAT1 or overexpressing miRNA-98-3p in ovarian cancer cell HEY-T30 in Example 2 on the angiogenesis ability and node formation of HUVEC;

[0036] Figure 14 Result diagram of the effect of knocking down MALAT1 or overexpressing miRNA-98-3p in ovarian cancer cell SKOV3 in Example 2 on the angiogenesis ability and node formation of HUVEC;

[0037] Figure 15 miRNA-98-3p inhibitor can rescue the inhibitory effect of knocking down MALAT1 on angiogenesis;

[0038] Figure 16 In ovarian cancer cells HEY-T30 and SKOV3, inhibiting miRNA-98-3p can rescue the inhibitory effect of knocking down MALAT1 on VEGFA expression, Western blot experimental result;

[0039] Figure 17 Schematic diagram of xenograft tumors;

[0040] Figure 18 Schematic diagram of the tumor weight and volume of mice treated with different groups;

[0041] Figure 19 Schematic diagram of the red pixels of xenograft tumors of mice treated with different groups;

[0042] Figure 20 Western blot results of protein levels of VEGFA, GAPDH, MMP2 and MMP9 in mice treated with different groups

[0043] Figure 21 Results graphs and data graphs of blood perfusion volume of different groups observed by laser speckle blood flow imaging system

[0044] Figure 22 Results graphs of immunohistochemical staining of mice treated with different groups Detailed implementation manners

[0045] The specific examples listed in the present invention are only used as examples of the present invention, and the present invention is not limited to the specific examples described below. For those skilled in the art, any equivalent modifications and substitutions to the examples described below are also within the scope of the present invention. Therefore, equivalent transformations and modifications made without departing from the spirit and scope of the present invention should all be covered within the scope of the present invention. Those not specified in the examples are carried out under conventional conditions or conditions recommended by the manufacturer. All reagents or instruments not specified by the manufacturer can be obtained through commercially available conventional products. In order to better illustrate the present invention, numerous specific details are given in the following detailed implementation manners. Those skilled in the art should understand that the present invention can also be implemented without some specific details. In other embodiments, methods, means, equipment and steps well known to those skilled in the art are not described in detail to highlight the gist of the present invention.

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Unless otherwise specified, the units used in this specification are all international standard units, and the numerical values and numerical ranges appearing in the present invention should be understood to include the inevitable systematic errors in industrial production.

[0047] The features and properties of the present invention will be further described in detail below in conjunction with the examples.

[0048] Example 1 Screening process of miRNA-98-3p

[0049] 1. In ovarian cancer, miRNA-98-3p is the direct targeting site of MALAT1

[0050] In ovarian cancer cell HEY-T30, the expression of MALAT1 was knocked down by two si-MALAT1 (si-MALAT1-1, si-MALAT1-2). As Figure 3The real-time RT-qPCR results shown indicate that two si-MALAT1 can effectively knockdown MALAT1 in HEY-T30 cells and can increase the expression levels of miR-98-3p, miR-4667-3p, and miR-6886-3p. The sequences of the two si-MALAT1 and Si-control are shown in Table 1.

[0051] Table 1. Primer sequences used in this example

[0052]

[0053] Subsequently, RNA-seq analysis was performed on the ovarian cancer cell line HEY-T30 after knockdown. The analysis results are as Figure 1 shown in Figure 1 Panel A. As can be seen in Panel A, after knockdown of MALAT1, the expression levels of miR-3611, miR-98, miR-8066, miR-4667, miR-450b, miR-384, and miR-6886 all changed.

[0054] Subsequently, the binding sites between these altered miRNAs and MALAT1 were predicted on the TargetScan website. The predicted binding sites are shown in Figure 1 Panel B. According to the predicted binding sites, MALAT1 was mutated in regions and a dual-luciferase reporter gene assay was performed. The results are as Figure 2 shown. Figure 2 It shows that the binding of miR-3611, miR-98-3p, miR-4667-3p, and miR-6886-3p to MALAT1 can all reduce the fluorescence intensity. Figure 3 It is shown that by performing qRT-qPCR experiments, compared with the si-control, si-MALAT1-1 and si-MALAT1-2 can effectively knockdown MALAT1 in HEY-T30 cells and can increase the expression levels of miR-98-3p, miR-4667-3p, and miR-6886-3p, and the knockdown effect of miR-98-3p is more stable.

[0055] These data demonstrate the direct targeting effect of MALAT1 on miRNA-98-3p in ovarian cancer, acting as a competing endogenous RNA (ceRNA) for each other.

[0056] 2. Knockdown of MALAT1 in ovarian cancer cells leads to a decrease in VEGFA expression

[0057] To further explore the role of the ceRNA mechanism formed by MALAT1 and miRNA-98-3p in ovarian cancer, MALAT1 was knocked down in HEY-T30 cells for RNA-seq to detect target genes. The results are shown asFigure 4 After knocking down MALAT1, the expression of VEGFA changed significantly. We speculated that the mechanism might be related to tumor angiogenesis associated with VEGFA. We detected a positive correlation between the expressions of MALAT1 and VEGFA in the clinical database of ovarian cancer, and as Figure 5 shown by the Kaplan-Meier Plotter data, higher levels of MALAT1 and VEGFA expressions were significantly associated with shorter survival time in ovarian cancer. Figure 6 The results also showed that knocking down MALAT1 inhibited the mRNA and protein expressions of VEGFA. This suggested that the mechanism of action of MALAT1 and miRNA-98-3p in ovarian cancer might be related to VEGFA.

[0058] Example 2 Verification process of miRNA-98-3p

[0059] 1. VEGFA is a direct targeting site of miRNA-98-3p in ovarian cancer cells

[0060] To further investigate the relationship and role of the mechanisms of action of MALAT1 and miRNA-98-3p in ovarian cancer with VEGFA, TargetScan was used to predict the possible interaction between miRNA-98-3p and VEGFA. The prediction results were as Figure 7 shown. The results indicated that VEGFA contains a miRNA-98-3p binding site. Next, a dual-luciferase reporter gene assay was used, and a reporter vector containing the miRNA-98-3p binding region in wild-type VEGFA 3'UTR and mutant VEGFA 3'UTR (sequence as Figure 7 the third sequence) was used to measure the relative luciferase activity. In HEK-293T cells co-transfected with miRNA-98-3p mimic and wild-type VEGFA 3'UTR, the relative luciferase activity decreased, as Figure 8 shown. The co-transfection method was as follows: ① Dilute 25 μL of medium to a final concentration of 500 ng plasmid (WT / MUT) + 80 nM mimic / Control; ② Dilute 1 μL of Lipo RNAiMAX reagent with 25 μL of medium. Let it stand at room temperature for 5 minutes, mix ① and ②, gently mix well, and incubate at room temperature for 5 minutes. After replacing the HEK-293T cells with 450 μL of basal medium, gently add 50 μL of the above mixture to each well. In addition, as Figure 9As shown, miRNA at a concentration of 150 nM (the amount used per well of a six-well plate) was added to ovarian cancer cells HEY-T30 and SKOV3, and miRNA-98-3p mimics, miRNA-98-3p inhibitors, and their negative controls (NC mimics and NC inhibitors) were transfected respectively (in the following steps, transfection was also carried out at this concentration without special instructions). The miRNA-98-3p mimics significantly reduced the mRNA expression level of VEGFA, and the miRNA-98-3p inhibitors significantly increased the mRNA expression level of VEGFA. The research results showed that there was a negative regulatory effect between miRNA-98-3p and VEGFA in ovarian cancer cells HEY-T30 and SKOV3. In summary, our research results indicated that VEGFA was a target gene of miRNA-98-3p in ovarian cancer cells.

[0061] 2. Knockdown of MALAT1 or overexpression of miRNA-98-3p inhibits the proliferation, migration, and tube formation of HUVECs

[0062] In previous studies, we have confirmed that the mechanism of action of MALAT1 and miRNA-98-3p in ovarian cancer is related to VEGFA. Next, to directly detect the effects of MALAT1 and miRNA-98-3p on angiogenesis, we performed several in vitro angiogenesis experiments using HUVECs.

[0063] After knocking down MALAT1 or overexpressing miRNA-98-3p in ovarian cancer cells HEY-T30 and SKOV3 respectively, the cell culture supernatant after centrifugation at low speed for 5 min was co-cultured with HUVEC cells for a cell proliferation experiment. The results were as Figure 10 shown. It was found that the cell culture supernatant of cells with knocked-down MALAT1 or overexpressed miRNA-98-3p both had a certain inhibitory effect on the growth of HUVEC cells. Similarly, as Figure 11 (the scale bar in the figure is 100 μm) and Figure 12 shown by the results of the scratch assay, compared with the negative control groups (si-NC and NC mimics), the cell culture supernatant after knocking down MALAT1 or overexpressing miRNA-98-3p could reduce the migration ability of HUVECs. As Figure 13 and Figure 14 shown, the cell culture supernatant of cells with knocked-down MALAT1 or overexpressed miRNA-98-3p could effectively inhibit the angiogenesis and node formation ability of HUVECs. These results indicated that knocking down MALAT1 or overexpressing miRNA-98-3p could inhibit the proliferation, migration, tube formation, and node formation of HUVECs.

[0064] 3. Simultaneous knockdown of MALAT1 and inhibition of miRNA-98-3p can reverse the expression of VEGFA and rescue angiogenesis in HUVECs

[0065] Based on the existing experimental results, we hypothesized that MALAT1 and miRNA-98-3p form a ceRNA to promote or inhibit tumor angiogenesis by regulating the target gene VEGFA. Next, to verify the relationship among MALAT1, miRNA-98-3p, and VEGFA, we conducted a rescue experiment. As Figure 15 (A, B) The angiogenesis assay showed that inhibition of miRNA-98-3p in ovarian cancer cells HEY-T30 and SKOV3 reversed the inhibitory effect of MALAT1 knockdown on angiogenesis. As Figure 16 The Western blot shown also confirmed our hypothesis and corroborated the previous experimental results. Simultaneous knockdown of MALAT1 and inhibition of miRNA-98-3p can reverse the expression of VEGFA and rescue angiogenesis in HUVECs. These data indicate that in ovarian cancer, MALAT1 and miRNA-98-3p form a ceRNA to affect tumor angiogenesis by regulating the target gene VEGFA.

[0066] 4. Overexpression of miRNA-98-3p can inhibit angiogenesis and tumor blood perfusion in a mouse model of ovarian cancer

[0067] We used HEY-T30 ovarian cancer cells with stable overexpression of miR-98-3p to conduct xenograft tumor experiments in nude mice bearing tumors, and observed the growth and angiogenesis of tumors in vivo. Each nude mouse was inoculated with 1*10^6 cells. The xenograft tumors are shown in Figure 17 and Figure 18 . Compared with the control group (inoculated with HEY-T30 ovarian cancer cells transfected with empty vector), the weight and volume of tumors in the miR-98-3p overexpression group were reduced. Compared with the control group, the tumors in the miR-98-3p overexpression group were paler. We quantified the degree of redness in the two groups of tumors, and the results are shown in Figure 19 , confirming that there were significantly more red pixels in the control group compared with the xenograft tumors in the miR-98-3p overexpression group. This is consistent with the previous in vitro research results. The Western blot results shown in Figure 20 indicated that overexpression of miRNA-98-3p in HEY-T30 ovarian cancer cells decreased the protein levels of VEGFA, MMP2, and MMP9. To analyze the tumor blood perfusion, the results are shown in Figure 21As shown, the blood perfusion volume in the miR-98-3p overexpression group was observed to be lower than that of the tumor in the control group by the laser speckle flow imaging system. To analyze tumor angiogenesis, tumor samples were evaluated by immunohistochemical staining with anti-CD31 antibody. The results are as Figure 22 shown. Compared with the control group, the miR-98-3p overexpression group showed a significant reduction in blood vessels. These results indicate that overexpression of miRNA-98-3p significantly inhibits tumor progression in vivo, and miRNA-98-3p regulates tumorigenesis by inhibiting VEGFA-mediated angiogenesis and blood perfusion volume.

[0068] The above-described embodiments only express the specific embodiments of the present application, and the description thereof is relatively specific and detailed, but it should not be construed as a limitation on the protection scope of the present application. It should be noted that for those of ordinary skill in the art, without departing from the concept of the technical solution of the present application, several modifications and improvements can be made, and these all belong to the protection scope of the present application.

Claims

1. The use of miRNA-98-3p in the preparation of anti-angiogenic drugs, characterized in that: The sequence of the miRNA-98-3p is: CUAUACAACUUACUACUUUCCC.

2. The use of miRNA-98-3p in the preparation of anti-angiogenic drugs according to claim 1, characterized in that: The miRNA-98-3p acts as a VEGFA inhibitor in anti-angiogenesis.

3. Application of miRNA-98-3p mimics in the preparation of anti-angiogenic drugs.

4. Application of miRNA-98-3p expression promoter in the preparation of anti-angiogenic drugs.

5. An anti-angiogenesis pharmaceutical composition, characterized in that: It includes miRNA-98-3p, miRNA-98-3p mimics or miRNA-98-3p expression promoter.

6. The anti-angiogenesis pharmaceutical composition according to claim 5, characterized in that: The single-use dosage of the miRNA-98-3p and miRNA-98-3p mimics is 150 nM.

7. The anti-angiogenesis pharmaceutical composition according to claim 5, characterized in that: The pharmaceutical composition also includes pharmaceutical excipients.

8. An anti-angiogenesis pharmaceutical composition according to claim 7, characterized in that: The auxiliary materials are sustained-release agents, fillers, adhesives, wetting agents, disintegrants, absorption promoters, adsorption carriers, surfactants or lubricants.

9. The anti-angiogenesis pharmaceutical composition according to claim 5, characterized in that: The dosage form of the pharmaceutical composition is aerosol, tablet, capsule, pill, powder, solution, suspension, emulsion, granule, lipid agent, lozenge, suppository or injection.

10. The anti-angiogenesis pharmaceutical composition according to claim 5, characterized in that: The miRNA-98-3p is natural, artificially synthesized, or a DNA fragment and expression vector expressing the same sequence as the miRNA-98-3p.

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