CN118634237A - Active ingredient of anti-oral tumor medicine and application thereof - Google Patents
Active ingredient of anti-oral tumor medicine and application thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/14—Type of nucleic acid interfering N.A.
- C12N2310/141—MicroRNAs, miRNAs
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Abstract
The invention discloses an active ingredient of a medicine for treating/preventing oral tumor and application thereof. The active ingredients can be derivatives of SEQ ID NO. 1-SEQ ID NO.6 and the like starting from SEQ ID NO. 1-SEQ ID NO.6, and can participate in regulating gene expression, so that the active ingredients have the effect of treating/preventing oral tumors; these active mirnas may regulate early development of immune cells, affecting immune cell development and differentiation; active miRNAs may be involved in the regulation of immune function, and have therapeutic/prophylactic effects on oral tumors. The transfection efficiency of the active miRNA in the liposome is high, and the anti-tumor effect after transfection is obvious.
Description
The application relates to a passive divisional application for overcoming singleness, the application number of the original application is 202210734741.7, the application date is 2022, 06 and 27, and the application name is 'active ingredient of an anti-oral tumor drug and application thereof'.
Technical Field
The invention belongs to the technical field of medicines, and relates to application of related active ingredients in preparation of medicines for treating/preventing oral tumors.
Background
Oral neoplasm (OralCancer) is a relatively common malignant neoplasm that severely endangers health and quality of life, and is highly localized in asia. According to the statistics made by GLOBOCAN in 2020, the total number of global cases of oral tumors was 37713, with asia population accounting for 65.8%, and the global mortality rate of oral tumor patients in 2020 was 177757, with asia accounting for 74%. Treatment of oral tumors mainly involves surgical excision, radiation therapy, chemotherapy, or a combination of several anticancer therapies. In recent years, although oral tumors have made a long-term progress in imaging diagnosis, surgical techniques, radiotherapy and chemotherapy, systemic therapy, etc., the survival rate of oral tumor patients within 5 years is still not ideal. Of particular concern, the survival of oral tumors has not been improved over the last three decades. One of the main reasons is that the first-line chemotherapeutic drugs for treating oral tumors induce drug resistance to tumors after use, and the drug-resistant oral tumors have stronger resistance to anticancer treatment, and the proliferation and invasion capacities of the drug-resistant oral tumors also rise, so that cancer foci infiltrate into adjacent tissues and even distant metastasis occurs. A combination of multiple drugs is often used clinically to overcome the resistance of tumor cells to a single drug. However, the emergence of tumor multidrug resistance significantly weakens the benefits of the combination therapeutic strategy. Therefore, the search for novel active ingredients with anti-oral tumor effect and the development of novel drugs are important for clinical treatment of oral tumor and prognosis of patients. miRNA is taken as one of candidate drug types for treating various human diseases including tumors, is an endogenous small RNA with the length of about 20-24 nucleotides, has various important regulation effects in human bodies, and is expected to relieve or overcome the drug resistance problem of troublesome tumors by adopting miRNA alone and in combination therapy. At present, related clinical researches are carried out, but the clinical researches are limited to a small number of cancer species such as lymphoma, melanin and the like.
Natural killer cells (NaturalKillercells, NK cells) derived from bone marrow lymphoid stem cells can induce cytolytic activity (without pre-sensitization or activation) of virus-infected cells and tumor cells. NK-92 is a human NK cell line whose growth and proliferation are dependent on IL-2, and NK-92MI is an IL-2 independent NK cell line derived from NK-92 cell line and obtained by gene transfection. Two humanized NK cell lines can be conveniently and economically amplified in large quantities for a long period of time, and have been proved to have cytotoxicity against many malignant tumors.
Extracellular vesicles (ExtracellularVesicles, EVs) are a collective term for various vesicles with membrane structures that are released by cells. Scientists were the earliest in 1983 to isolate extracellular vesicles carrying the parent cellular components from sheep reticulocytes and are widely present in body fluids. Because exosomes carry important biomolecules such as nucleic acids, lipids and the like, the exosomes have great clinical application potential in early diagnosis, prognosis and treatment of cancers. Furthermore, exosomes act as drug delivery vehicles for the transfer of mirnas and therapeutic agents to target cells, and these nanovesicles have higher safety and stability compared to synthetic vehicles, which offers the possibility of targeted drug delivery in cancer treatment.
The invention utilizes two humanized NK cell strains NK-92 and NK-92MI as sources for obtaining extracellular vesicles secreted by NK cells.
Disclosure of Invention
In view of the above background, an object of the present invention is to provide an active ingredient and its use in preparing a medicament for treating/preventing oral tumor.
Through researches, the invention provides the following technical scheme: an active ingredient for preparing an anti-oral tumor drug selected from one of the following:
(a) miRNA-X with a sequence shown in any one of SEQ ID NO. 1-SEQ ID NO.6, or any combination of the miRNA-X, or modified miRNA-X derivatives;
SEQ ID NO.1(bta-miR-2478-L-2):ATCCCACTTCTGACACCA；
SEQ ID NO.2(hsa-miR-1260a):ATCCCACCTCTGCCACCA；
SEQ ID NO.3(hsa-miR-197-3p):TTCACCACCTTCTCCACCCAGC；
SEQ ID NO.4(hsa-miR-296-5p):AGGGCCCCCCCTCAATCCTGT；
SEQ ID NO.5(hsa-miR-339-5p):TCCCTGTCCTCCAGGAGCTCACG；
SEQ ID NO.6(hsa-miR-223-3p):TGTCAGTTTGTCAAATACCCCA
(b) A precursor miRNA that is processable in a host into the miRNA-X described in (a);
(c) A polynucleotide capable of being transcribed by a host to form the precursor miRNA of (b) and processed to form the microrna of (a);
(d) An expression vector comprising a microrna of miRNA-X as described in (a), or a precursor miRNA as described in (b), or a polynucleotide as described in (c);
(e) An agonist of the microRNA described in (a).
Experimental study shows that the combined use of miRNA-X shown in SEQ ID NO.1 and SEQ ID NO.3 has more remarkable inhibition effect on oral tumor cells:
Specifically, the agonist is selected from the group consisting of: substances that promote expression of miRNA-X, and substances that increase miRNA-X activity.
The invention also provides application of the active ingredient, wherein the active ingredient is used for preparing a medicament for treating/preventing oral tumors. The medicine can also contain the active ingredients and a pharmaceutically acceptable carrier.
Specifically, the preparation form of the medicine is freeze-dried powder injection, micro-needle, injection, tablet, patch, capsule, oral suspension or microsphere for interventional embolism.
Specifically, the drug delivery method comprises the following steps: and the delivery modes of a transfer method, a drug loading method, a direct naked RNA injection method, a liposome coated RNA direct injection method, a nanomaterial assembly method and the like are used for delivering the cationic material compound.
The invention has the beneficial effects that: according to the invention, three-stage separation is carried out on NK-92 cells and extracellular vesicles secreted by NK-92MI cells for the first time, two groups of extracellular vesicles with large, medium and small sizes are obtained, and then active screening is carried out on a large sample consisting of the miRNA sequences of the NK-92 cells and the NK-92MI cells and the miRNA sequences of the two groups of extracellular vesicles, so that active miRNA with strong killing power on oral tumor cells is obtained. The active miRNA obtained by the invention can be involved in regulating gene expression, has certain advantages in the treatment/prevention of oral tumors, and particularly has more remarkable advantages when SEQ ID NO.1 and SEQ ID NO.3 are used in combination; these active mirnas may regulate early development of immune cells, affecting immune cell development and differentiation; active miRNAs may be involved in the regulation of immune function, and have therapeutic/prophylactic effects on oral tumors. The transfection efficiency of the active miRNA in the liposome is high, and the anti-tumor effect after transfection is obvious.
Drawings
FIG. 1 is a flow chart of the extraction of extracellular vesicles of different sizes from two cell lines NK-92 and NK-92 MI;
FIG. 2 electron microscopy images of different sizes of extracellular vesicles secreted by NK-92 and NK-92MI cell lines;
FIG. 3 particle size diagram of different size extracellular vesicles secreted by NK-92 and NK-92MI cell lines;
FIG. 4 graphs of the anti-oral tumor effects of NK-92 and NK-92MI cell lines;
FIG. 5 is a chart of the miRNA profile of NK-92 and NK-92MI cell lines;
FIG. 6 shows the anti-oral tumor effect of different sizes of extracellular vesicles secreted by NK-92 and NK-92MI cell lines, respectively;
FIG. 7 is a chart of the biological analysis of extracellular vesicle miRNAs of different sizes secreted by NK-92 and NK-92MI cell lines, respectively;
FIG. 8 shows the transfection effect of active miRNA-Xmimics (mimetic) and miRNA-Xinhibitors (inhibitor);
FIG. 9 is a graph of the effect of mimics (mimetic) of active miRNA-X on anti-oral tumors following transfection of miRNA-Xinhibitors (inhibitor);
Figure 10 graph of the effect of mimics (mimetic) of active miRNA-X against oral tumors after co-transfection.
Detailed Description
Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings. The experimental procedure, in which specific conditions are not specified in the preferred embodiments, is generally carried out according to conventional conditions, or according to the conditions recommended by the reagent manufacturers.
Example 1 active miRNA-X screening
In this example, all of the cell lines used were human cell lines, and all were purchased from the national academy of sciences typical culture preservation committee cell bank (NationalCollectionofAuthenticatedCellCultures, national model and specialty laboratory cell resource bank).
The culture solution used by the human immune cell strain NK-92 comprises the following components: MEM-alpha (Hyclone, UT, USA) with 12.5% horse serum (Gibco, CA, USA) and 12.5% fetal bovine serum (Gibco, CA, USA), and 0.2mM inositol (Sigma, DA, DE), 0.1mM beta mercaptoethanol (Sigma, DA, DE), 0.02mM folic acid (Sigma, DA, DE) and 200U/mL recombinant IL-2 (Novoprotein, SHH, CN) were added.
The culture medium of the human immune cell line NK-92MI has a similar composition to that of the above NK-92 cell line but does not contain recombinant IL-2.
Human oral tumor cell line KB was cultured in DMEM medium (Gibco, CA, USA) containing 10% normal fetal bovine serum and 1% penicillin-streptomycin (Yeasen, SHH, CN).
All cell lines were cultured in a cell incubator (ThermoFisherScientific, MA, USA) maintained at 37℃and containing 5% CO 2 and a humid environment.
1.1 Isolation, acquisition and purification of Extracellular Vesicles (EVs)
Exosome-free culture solution preparation: serum (containing horse serum and fetal bovine serum mixed in a ratio of 1:1) and MEM-alpha culture solution are mixed in a volume ratio of 1:4, and then are split into ultracentrifuge tubes (specification: 25X 89 mm) on average and balanced, the ultracentrifuge tubes are placed into an ultracentrifuge and centrifuged for 16h (ultracentrifuge rotor: SW32Ti, beckman, CA, USA) at a centrifugal force of 120000g under the condition of 4 ℃, and the supernatant collected after centrifugation is the culture solution without exosomes.
NK-92 and NK-92MI cell lines were cultured in the above Exosome-free medium (EVs-free medium) for 2-3 days, respectively, followed by the following three experimental procedures, respectively, to obtain extracellular vesicles LEV (LargeExtracellularVesicle), extracellular vesicles EXO (exosomes), and extracellular vesicles SEV (SmallExtracellularVesicle), respectively.
1.1.1 Isolation and acquisition of extracellular vesicles LEV: ① Respectively collecting culture solutions of two strains of cells after 2-3 days of culture, centrifuging at 4deg.C for 10min with centrifugal force of 400g, and collecting supernatant; ② Centrifuging the supernatant at 4deg.C with centrifugal force of 2000g for 20min, discarding the bottom precipitate (which is cell and debris), and collecting supernatant; ③ Centrifuging the obtained supernatant at 4deg.C for 30min with 10000g centrifugal force, centrifuging the precipitate with Phosphate Buffer (PBS) at 4deg.C for 30min with 10000g centrifugal force to wash the precipitate, and collecting the precipitate respectively as LEVs derived from two immunocyte strains, namely NK-92LEV and NK-92MILEV (shown in figure 2; figure 3); ④ The LEV pellet was gently beaten, dissolved and homogenized with 50-100 mu LPBS, respectively, and stored at-80 ℃. 1.1.2 isolation and acquisition of extracellular vesicles EXO: ① Filtering supernatant obtained in experimental operation ③ by centrifugation at 10000g under 4deg.C for 30min with sterile filter with diameter of 0.22 μm, and collecting the filtered liquids; ② Centrifuging the filtered collected liquids at a centrifugal force of 110000g at 4deg.C for 70min (super-centrifugal rotor: SW32 Ti), respectively, and collecting the obtained precipitates; ③ Re-washing the obtained precipitate with PBS, centrifuging and washing at 110000g at 4deg.C for 70min, and repeating the experimental operation of PBS re-suspension and washing for 1-2 times to obtain precipitate as EXO derived from two immune cell lines, namely NK-92EXO and NK-92MIEXO (shown in figure 2; figure 3); ④ The EXO precipitate was gently beaten, dissolved and mixed with 50-100 mu LPBS, respectively, and stored at-80 ℃.
1.1.3 Isolation and acquisition of extracellular vesicles SEV: ① Centrifuging the liquid collected after filtration in experiment operation ① of 1.1.2 at 4deg.C for 70min at 110000g, and collecting supernatant while collecting precipitate in experiment operation ② of 1.1.2 for separating and obtaining extracellular vesicles EXO; ② Centrifuging the obtained supernatant at a rotation speed of 167000g for 16h at 4 ℃ respectively, and collecting the obtained precipitate respectively; ③ Re-washing the obtained precipitate with PBS, centrifuging and washing for 4 hr at 4deg.C with centrifugal force 167000g, and repeating the experimental operation of PBS re-suspension and washing for 2-3 times to obtain precipitate as SEV derived from two immunocyte strains, namely NK-92SEV and NK-92MISEV (shown in figure 2; figure 3); ④ The EXO precipitate was gently beaten, dissolved and mixed with 50-100 mu LPBS, respectively, and stored at-80 ℃.
The flow of separating and obtaining the different types of extracellular vesicles is summarized as shown in figure 1, and the transmission electron microscope and the particle size characterization of the obtained extracellular vesicles are respectively shown in figures 2 and 3.
After two groups of 6 extracellular vesicles were isolated and obtained by the above experimental method, the RNA adsorbed on the vesicle surface was further removed by the following experimental procedure: the obtained EVs were resuspended in PBS, respectively, and an appropriate amount of RNase (RNaseA, ambion, TX, USA) was added to give a final concentration of RNaseA of 1U/mL, followed by incubating the RNaseA-containing extracellular vesicle solutions in a 37℃water bath (JingHong, SHH, CN) for 20min. This experimental procedure was operated following the guidelines of the International Association of extracellular vesicles (InternationalSocietyforExtracellularVesicles, ISEV). 1.2 sequencing and bioinformatic analysis of miRNAs of human cell lines NK-92 and NK-92MI and their secreted extracellular vesicles
MiRNA sequencing of 1.2.1NK-92 and NK-92MI cell lines
We extracted total RNA (including all miRNA and small molecule RNA) from NK-92 and NK-92MI cultured cell samples, respectively, using a total RNA purification kit (NorgenBiotekCorp, thorold, ON, canada); the quality and quantity of the obtained RNA was analyzed using a Bioanalyzer2100 (Agilent, calif., USA) and an RIN value >7.0 was ensured; taking 1 mug total RNA from RNA samples extracted from two cell strains respectively, and preparing a small molecular RNA library by using a TruSeq small molecular RNA sample preparation kit (Illumina, SD, USA); the 50bp single-ended sequencing of miRNAs in the two cell line samples was performed by the company Lithospermum Co., ltd (LCSCIENCES, HZ, CN) according to the instructions provided by the manufacturer of IlluminaHiSeq2500 (Hanyu, SHH, china).
1.2.2 MiRNA sequencing of two groups of extracellular vesicles
To remove the RNase added in 1.1 and to simultaneously purify the extracellular vesicles, we re-performed the super-isolation and washing according to the procedure of obtaining and purifying the corresponding extracellular vesicles in 1.1; extracting total RNA of various extracellular vesicles by adopting the same method as in 1.2.1, preparing a small molecular RNA library, and respectively carrying out 50bp single-ended sequencing on miRNA in various extracellular vesicle samples.
1.2.2.3 Bioinformatic analysis of sequencing results of miRNAs
First, bioinformatics analysis was performed on miRNA sequencing data of human cell lines and secreted extracellular vesicles using ACGT101-miR analysis software (LCSCIENCES, TX, USA). The main analysis flow of the software is as follows: carrying out quality control treatment on the original data to obtain cleanreads, removing the 3' joint from CLEAN READS, carrying out length screening, and reserving a sequence with the base length of 18-26 nt; the remaining sequences are then aligned with RNA database sequences, such as mRNA database, RFam database (including rRNA, tRNA, snRNA, snoRNA, etc.), and Repbase database (repeat database), and filtered.
Secondly, miRNAs with potentially important biological functions are screened based on sequencing data and bioinformatics analysis software. The main screening method and the process are as follows: ① Based on DESeq2 (V1.26.0) (R language software package), differential expression analysis is carried out on the miRNA sequencing results of two cell strains and the miRNA sequencing results of two groups of extracellular vesicles, miRNAs which accord with log 2 FoldChange >1.25 and the statistical analysis p value <0.05 are taken as differential expression miRNAs and cluster analysis is carried out, 15 miRNAs with high expression and significant difference (NK 92 is significantly down-regulated compared with NK 92-MI) were screened from the sequencing data of the two cell lines (FIG. 5 and Table 1), and 50 miRNAs with high co-expression were screened from the sequencing data of the extracellular vesicles (FIG. 7 and Table 2); ② Based on the assumption that the miRNA with functions has advantages on the expression level, calculating the average expression amounts of the miRNA of the cell strain and the extracellular vesicles respectively, and sequencing the differentially expressed miRNA based on the average expression amounts; ③ According to the expression quantity and the change times of the miRNAs screened by ①, finally, 4 target miRNAs which are obviously higher than NK-92 in NK-92MI are screened from the miRNAs obtained by preliminary screening of the 15 cell strains (the experiment research shows that the anti-oral tumor effect of the NK-92MI cell strain is superior to that of the NK-92 cell strain, The results are shown in FIG. 4, analyzed using GraphPadprism software One-wayANOVA; *,P<0.05,** P < 0.01), including 1 unknown miRNA (bta-miR-2478_L-2) (fold difference most pronounced) and 3 known miRNAs (hsa-miR-1260 a, hsa-miR-197-3P, hsa-miR-296-5P) (FIG. 5). Meanwhile, 2 target miRNAs with expression significantly higher than LEV and SEV in EXO are selected from miRNAs obtained by preliminary screening of the 50 extracellular vesicle miRNAs (we find that EXO has significantly better anti-oral tumor effect than LEV and SEV through experimental study, and the result is shown in figure 6, and the analysis is carried out by using GraphPadprism software One-wayANOVA; *,P<0.05,**,P<0.01,*** P < 0.001), including the known hsa-miR-339-5P and hsa-miR-223-3P (FIG. 7).
The 6 marked miRNA is the active miRNA, and is marked as miRNA-X.
TABLE 1 sequencing results of the first 15 miRNAs with significant changes in expression levels between NK-92/NK-92MI cell lines
TABLE 2 sequencing results of 50 miRNAs with high EVs Co-expression in NK-92/NK-92MI
EXAMPLE 2 construction of active miRNA-X expression vector
2.1 Construction of active miRNA-X Single expression vector
In this example, lipofectamine3000 liposome was used for expression vector construction, the concentrations of miRNA-Xmimic and mimic controls were set at 60nM, and experiments were performed in 96-well plates with a total system per well of 200. Mu.L. The specific operation is as follows:
① 0.6 mu LmiRNA-Xmimic and equal amounts of mimicNC (NC is a nematode miRNA sequence) were added to 9.4 mu Lopti-MEM, respectively;
② Adding 0.3 mu LLipofectamine to 9.7 mu Lopti-MEM, and standing for 5min;
③ ① is added into ②, and the mixture is kept stand for 15min after being lightly blown, so as to obtain the Lipofectamine3000 liposome expressing miRNA-X and the Lipofectamine3000 liposome expressing mimicNC.
2.2 Construction of active miRNA-X Combined expression vector
The implementation adopts Lipofectamine300 liposome to construct a combined expression vector, the concentration of miRNA-Xmimic and mimic contrast is set to be 60nM, and the experiment is carried out in a 96-well plate, and the total system of each well is 200 mu L. The specific operation is as follows:
① 0.6 mu LmiRNA-X1mimics, miRNA-X2mimics and mimicNC, respectively, was added to 10 mu Lopti-MEM. Wherein mimicNC prepares two parts by using the same formula and method;
② Two liposome solutions were prepared: 0.6 mu LLipofectamine of 3000 was added to 20 mu Lopti-MEM, and the mixture was left to stand for 5 minutes after the addition. Preparing two liposome solutions with the same formula and method;
③ One part of liposome solution is added with the miRNA-X1mimics and miRNA-X2mimics solution prepared in ①, and the other part of liposome solution is added with mimicNC prepared in ①; after gently blowing, the mixture was allowed to stand for 15 minutes.
The miRNA-X expression vectors and miRNA-X1mimics and miRNA-X2mimics expression vectors obtained by constructing the 2.1 and 2.2 are actually representative dosage forms for miRNA application, and provide a thinking for the subsequent preparation of various medicines based on single or combined use of active miRNA-Xmimics. The liposome medicine containing the one or more active miRNA-Xmimics combinations and the activator/inhibitor thereof can be prepared later, and the administration mode is external application or injection.
Example 3 active miRNA-X transfection efficiency test
To verify the transfection efficiency of the 6 mirnas-X screened in example 1 in oral tumor cells, we performed in vitro transfection experiments. Firstly, 6 miRNA-Xmimics (RiboLifeScience, JS, CN) with the same active miRNA-X screened in the embodiment 1 are selected to be incubated with a human oral tumor cell strain KB, and the specific experimental steps are as follows:
① Inoculating KB cells: KB cells in the logarithmic growth phase were seeded at 1X 10 5～5×105 cells/well in 6-well plates.
② Lipofectamine3000 liposomes expressing miRNA-X obtained by the construction of example 2.1 were added to KB cells.
③ After 4h of transfection, the liquid is changed, KB cells are collected after the culture is continued for 48h, and the transfection efficiency is detected by using an RT-PCR experiment.
The expression level of active miRNA-X in KB oral tumor cells was detected by RT-PCR experiments, and we confirmed that the expression level of 6 miRNA-X was significantly increased after transfection of miRNA-Xmimics (FIG. 8).
To further verify the transfection efficiency of the 6 inhibitors of miRNA-X screened in example 1, we incubated the corresponding 6 mirnas-Xinhibitors (RiboLifeScience, JS, CN) with human oral tumor cell line KB, with the following specific experimental steps: KB cells in the logarithmic growth phase were seeded at 1X 10 5～5×105 cells/well into 6-well plates; the concentrations of miRNA-Xinhibitor and inhibitor controls were set at 120nM, and the specific procedures were as follows:
① Adding 12 mu LmiRNA-Xinhibitor and inhibitor control into 88 mu Lopti-MEM respectively;
② Adding 7.2 mu LLipofectamine to 92.8 mu Lopti-MEM, and standing for 5min;
③ ① was then added to ② and left to stand for 15min after gentle blowing. Then added to KB cells. Changing the liquid after 4 hours of transfection, continuously culturing for 48 hours, collecting KB cells, and detecting the transfection efficiency by using an RT-PCR experiment;
The expression level of active miRNA-X in KB oral tumor cells was detected by RT-PCR experiments, and we confirmed that the expression level of 6 miRNA-X was significantly reduced after transfection of miRNA-Xinhibitor (FIG. 8).
From the above miRNA-Xmimics and inhibition transfection experiments, it can be seen that: the miRNA-X obtained by screening is successfully transfected into oral tumor cells by the vector system constructed by the method, and the expression of the miRNA-X in the tumor cells respectively generates a regulating effect which meets expectations and ideal.
Example 4 anti-oral tumor Activity test of active miRNA-X
To further verify that miRNA-X has an anti-oral tumor effect, we incubated active miRNA-Xmimics/inhibitors with KB human oral tumor cell lines. The specific experimental steps are as follows:
Transfecting miRNA-Xmimics/inhibitors into KB human luminal tumor cells, the transfection procedure being as described in example 3; after 4h transfection, the transfection system is changed with liquid, and after 48h culture is continued, 3- (4, 5-dimethylthiazole-2) -2, 5-diphenyl tetrazolium bromide (MTT, sigma, DA, DE for short) is added to detect the survival rate of KB cells, and the method is used for evaluating the antitumor effect of miRNA-Xmimics/inhibitors on KB cells.
Experimental results show that 6 miRNAs-Xmimics have a remarkable effect of inhibiting tumor proliferation on KB oral tumor cells, while miRNA-Xinhibitors has no effect of inhibiting KB tumor cell proliferation (FIG. 9, analyzed by GraphPadprism software One-wayANOVA. **,P<0.01;***, P < 0.001.).
Therefore, any one of 6 miRNA-X is over-expressed, so that proliferation of oral tumor cells can be obviously inhibited, and an anti-oral tumor effect can be realized. Those skilled in the art can expect that the active ingredients developed based on 6 mirnas-X can also significantly inhibit proliferation of oral tumor cells and exert anti-oral tumor effects, including but not limited to: ① miRNA-X derivatives modified by miRNA-X; ② Precursor miRNAs which can be processed into the 6 miRNAs-X in a host; ③ A polynucleotide capable of being transcribed by a host to form a precursor miRNA described in ②; ④ An expression vector comprising a miRNA-X or a miRNA-X derivative as described in ①, or an expression vector of a precursor miRNA as described in ②, or an expression vector of a polynucleotide as described in ③ (example 2); ⑤ Agonists that promote miRNA-X expression and/or increase miRNA-X activity.
Example 5 anti-oral tumor Activity test of active miRNA-X combination therapy
In order to further verify that miRNA-X combined treatment has the effect of resisting oral tumors, the combined expression vector constructed in 2.2 is incubated with a human oral tumor cell strain KB, and the specific experimental steps are as follows:
① Inoculating KB cells: KB cells in the logarithmic growth phase were seeded at 1X 10 3～5×103 cells/well into 96-well plates;
② Lipofectamine3000 liposome expressing miRNA-X1mimics and miRNA-X2mimics obtained by constructing in example 2 is added into KB cells;
③ After culturing for 48h, 3- (4, 5-dimethylthiazole-2) -2, 5-diphenyl tetrazolium bromide (MTT for short) is added to detect the survival rate of KB cells, and the method is used for evaluating the anti-tumor effect of miRNA-Xmimics combined treatment on KB cells.
Experimental results show that the combination of different miRNA-X can generate good inhibition/killing effect on KB cells. For example, there were statistically significant differences in cancer cell survival rates for the three miRNA-combined groups versus the NC control group (fig. 10, P <0.05;, P <0.01;, P < 0.001) analyzed using GraphPadPrism software One-wayANOVA. On the other hand, the anti-oral cancer effect of different miRNA-X combination is different, and the specific combination can inhibit/kill KB cells more effectively. For example, when bta-miR-2478-L-2 is combined with hsa-miR-197-3p, compared with the bta-miR-2478-L-2 and hsa-miR-197-3p which are respectively used independently, the anti-oral cancer activity is remarkably improved: 100.98% (P < 0.001) higher than bta-miR-2478-L-2 alone, 38.12% (P < 0.01) higher than hsa-miR-197-3P alone.
Unlike the combination effect of bta-miR-2478-L-2 and hsa-miR-197-3P, the combination effect of bta-miR-2478-L-2 and hsa-miR-339-5P is higher than the single anti-cancer activity (P < 0.05) of hsa-miR-339-5P, and is similar to the single anti-cancer activity of bta-miR-2478-L-2; the anti-cancer activity of the combination of hsa-miR-339-5P and hsa-miR-223-3P is higher than that of the single hsa-miR-339-5P (P is less than 0.05), and is similar to that of the single hsa-miR-223-3P.
The degree of enhancement or attenuation of the above anticancer activity is calculated by the following formula: (average anticancer effect of single use group-average anticancer effect of combined use group)/average anticancer effect of single use group X100%.
Finally, it is noted that the above-mentioned preferred embodiments are only intended to illustrate rather than limit the invention, and that, although the invention has been described in detail by means of the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention as defined by the appended claims.
Claims (6)
1. An active ingredient for use in a medicament for the treatment/prophylaxis of oral neoplasms, said active ingredient being selected from at least one of the following:
(a) miRNA-X with a sequence shown as SEQ ID NO.6 or a derivative of modified miRNA-X;
(b) A precursor miRNA that is processable in a host into the miRNA-X described in (a);
(c) A polynucleotide capable of being transcribed by a host to form a precursor miRNA as described in (b) and processed to form a miRNA as described in (a);
(d) An expression vector comprising a miRNA of miRNA-X as set forth in (a), or a precursor miRNA as set forth in (b), or a polynucleotide as set forth in (c);
(e) An agonist of the miRNA described in (a).
2. The active ingredient of claim 1, wherein the agonist is selected from the group consisting of: substances that promote expression of miRNA-X, and substances that increase miRNA-X activity.
3. Use of an active ingredient according to claim 1 or 2 for the preparation of a medicament for the treatment/prophylaxis of oral tumors.
4. The use according to claim 3, wherein the medicament comprises an active ingredient according to claim 1 or 2, and a pharmaceutically acceptable vehicle or carrier.
5. The use according to claim 3, wherein the pharmaceutical preparation is in the form of lyophilized powder for injection, microneedle, injection, tablet, patch, capsule, oral suspension or microsphere for interventional embolism.
6. The use according to claim 3, wherein the method of drug delivery comprises: a transfer method, a drug loading method, a direct naked RNA injection method, a liposome coated RNA direct injection method, a nano material assembly method and other cationic material complex delivery methods, and a bacterial plasmid expression RNA method, a virus package expression RNA method and other delivery modes.
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