CN118634241A - Active ingredients of anti-oral tumor drugs and their uses - Google Patents

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

Active ingredients of anti-oral tumor drugs and their uses

This application is a passive divisional application to overcome unity. The application number of the original application is 202210734741.7, the application date is June 27, 2022, and the name of the invention is "Active ingredients of anti-oral tumor drugs and their uses".

Technical Field

The present invention belongs to the field of medical technology, and relates to the use of relevant active ingredients in preparing drugs for treating/preventing oral tumors.

Background Art

Oral cancer is a common malignant tumor that seriously threatens health and quality of life. It is highly prevalent in Asia. According to statistics from GLOBOCAN in 2020, there were 37,713 new cases of oral cancer worldwide, of which 65.8% were in Asia. In 2020, the global mortality rate of oral cancer patients was 177,757, of which 74% were in Asia. The treatment of oral cancer mainly includes surgical resection, radiotherapy, chemotherapy, or a combination of several anticancer therapies. In recent years, although oral cancer has made great progress in imaging diagnosis, surgical techniques, radiotherapy, chemotherapy, and systemic therapy, the 5-year survival rate of oral cancer patients is still not ideal. It is particularly noteworthy that the survival rate of oral cancer has not improved in the past three decades. One of the main reasons is that the first-line chemotherapy drugs used to treat oral cancer will induce drug resistance in the tumor after use. Resistant oral tumors not only have stronger resistance to anticancer treatment, but also increase their ability to proliferate and invade, leading to the infiltration of cancer foci into adjacent tissues and even distant metastasis. In clinical practice, multi-drug combination therapy is often used to overcome the resistance of tumor cells to a single drug. However, the emergence of tumor multidrug resistance significantly weakens the benefits of combined treatment strategies. Therefore, finding new active ingredients with anti-oral tumor effects and developing new drugs are crucial to the clinical treatment of oral tumors and patient prognosis. As one of the candidate drug types for the treatment of various human diseases including tumors, miRNA is a class of endogenous small RNAs with a length of approximately 20-24 nucleotides. It has a variety of important regulatory effects in the human body. The use of miRNA alone and in combination therapy is expected to alleviate or overcome the difficult problem of tumor resistance. At present, relevant clinical studies have been conducted, but they are limited to a small number of cancers such as lymphoma and melanoma.

Natural Killer cells (NK cells) are derived from bone marrow lymphoid stem cells and can induce cytolytic activity of virus-infected cells and tumor cells (without prior sensitization or activation). NK-92 is a human-derived NK cell line that depends on IL-2 for growth and proliferation, and NK-92MI is an IL-2-independent NK cell line derived from the NK-92 cell line and obtained by gene transfection. The two human-derived NK cell lines can easily and economically achieve stable, large-scale, and long-term expansion, and have been proven to be cytotoxic to many malignant tumors.

Extracellular vesicles (EVs) are a general term for various membrane-structured vesicles released by cells. Scientists first isolated extracellular vesicles carrying parent cell components from sheep reticulocytes in 1983, and they are widely present in body fluids. Since exosomes carry important biological molecules such as nucleic acids and lipids, they have great clinical application potential in the early diagnosis, prognosis and treatment of cancer. In addition, exosomes are used as drug delivery carriers for the transfer of miRNA and therapeutic agents to target cells. Compared with synthetic carriers, these nanovesicles have higher safety and stability, which provides the possibility for targeted drug delivery in cancer treatment.

The present invention utilizes two human-derived NK cell strains, NK-92 and NK-92MI, as sources for obtaining extracellular vesicles secreted by NK cells.

Summary of the invention

In view of the above background technology, the object of the present invention is to provide an active ingredient and its use in preparing a drug for treating/preventing oral tumors.

After research, the present invention provides the following technical solution: an active ingredient for preparing an anti-oral tumor drug, selected from one of the following:

(a) a miRNA-X having a sequence as shown in any one of SEQ ID NO.1 to SEQ ID NO.6, or any combination of said miRNA-X, or a modified miRNA-X derivative;

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, wherein the precursor miRNA can be processed into the miRNA-X described in (a) in the host;

(c) a polynucleotide, which can be transcribed by a host to form the precursor miRNA described in (b), and processed to form the microRNA described in (a);

(d) an expression vector comprising the microRNA of miRNA-X described in (a), the precursor miRNA described in (b), or the polynucleotide described in (c);

(e) An agonist of the microRNA described in (a).

Experimental studies have found that the combined use of miRNA-X shown in SEQ ID NO.1 and SEQ ID NO.3 has a more significant inhibitory effect on oral tumor cells:

Specifically, the agonist is selected from the following group: a substance that promotes the expression of miRNA-X, a substance that increases the activity of miRNA-X.

The present invention also provides a use of the above active ingredient, wherein the active ingredient is used to prepare a drug for treating/preventing oral tumors. The drug may also contain the above active ingredient and a pharmaceutically acceptable carrier.

Specifically, the drug is in the form of freeze-dried powder injection, microneedle, injection, tablet, patch, capsule, oral suspension, or microspheres for interventional embolization.

Specifically, the drug delivery methods include: cationic material complex delivery such as transfection method, drug loading method, direct naked RNA injection method, liposome-encapsulated RNA direct injection method and nanomaterial assembly method, as well as bacteria carrying plasmids to express RNA method, virus packaging expression RNA method and other delivery methods.

The beneficial effects of the present invention are as follows: the present invention performs three-stage separation of the extracellular vesicles secreted by NK-92 cells and NK-92MI cells for the first time, obtains two groups of extracellular vesicles including three sizes of large, medium and small, and then uses the miRNA sequences of NK-92 cells and NK-92MI cells and the large sample composed of the miRNA sequences of the two groups of extracellular vesicles for activity screening, and obtains active miRNA with strong killing power against oral tumor cells. The active miRNA obtained by the present invention can participate in regulating gene expression, and has certain advantages in the treatment/prevention of oral tumors, especially when SEQ ID NO.1 and SEQ ID NO.3 are used in combination; these active miRNAs may regulate the early development of immune cells, affect the development and differentiation of immune cells; active miRNAs may participate in the regulation of immune function, and have a therapeutic/preventive effect on oral tumors. The active miRNA proposed by the present invention has a high transfection efficiency in liposomes, and has a significant anti-tumor effect after transfection.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 Flow chart of extracting extracellular vesicles of different sizes from NK-92 and NK-92MI cell lines;

Fig. 2 Electron microscopic images of extracellular vesicles of different sizes secreted by NK-92 and NK-92MI cell lines;

FIG3 is a graph showing the particle sizes of extracellular vesicles of different sizes secreted by NK-92 and NK-92MI cell lines;

Fig. 4 shows the anti-oral tumor effects of NK-92 and NK-92MI cell lines;

Figure 5 miRNA bioinformatics analysis of NK-92 and NK-92MI cell lines;

Figure 6 shows the anti-oral tumor effects of extracellular vesicles of different sizes secreted by NK-92 and NK-92MI cell lines;

Figure 7 Bioinformatics analysis of miRNAs in extracellular vesicles of different sizes secreted by NK-92 and NK-92MI cell lines;

Figure 8 shows the transfection effects of active miRNA-Xmimics (mimics) and miRNA-Xinhibitors (inhibitors);

FIG9 shows the anti-oral tumor effect of active miRNA-X mimics (mimics) and miRNA-X inhibitors (inhibitors) after transfection;

FIG. 10 shows the anti-oral tumor effect after co-transfection of active miRNA-X mimics.

DETAILED DESCRIPTION

The preferred embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings. The experimental methods in the preferred embodiments without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the reagent manufacturer.

Example 1. Screening of active miRNA-X

In this example, the cell lines used are all human cell lines, all purchased from the Cell Bank of the Committee for the Preservation of Type Cultures of the Chinese Academy of Sciences (National Collection of Authenticated Cell Cultures, National Model and Characteristic Experimental Cell Resource Bank).

The culture medium used for the human immune cell line NK-92 consists of MEM-α (Hyclone, UT, USA) supplemented with 12.5% horse serum (Gibco, CA, USA) and 12.5% fetal bovine serum (Gibco, CA, USA), as well as 0.2 mM inositol (Sigma, DA, DE), 0.1 mM β-mercaptoethanol (Sigma, DA, DE), 0.02 mM folic acid (Sigma, DA, DE) and 200 U/mL recombinant IL-2 (Novoprotein, SHH, CN).

The composition of the culture medium of the human immune cell line NK-92MI is similar to that of the culture medium of the above-mentioned 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 culture incubator (Thermo Fisher Scientific, MA, USA) maintained at 37°C with 5% CO ₂ and a humidified environment.

1.1 Isolation, acquisition and purification of extracellular vesicles (EVs)

Preparation of exosome-free culture medium: serum (containing 1:1 mixed horse serum and fetal bovine serum) and MEM-α culture medium were mixed in a volume ratio of 1:4, and evenly distributed into ultracentrifuge tubes (specifications: 25×89 mm) and balanced. The ultracentrifuge tubes were placed in an ultracentrifuge and centrifuged at 120,000 g for 16 h at 4°C (ultracentrifuge rotor: SW32Ti, Beckman, CA, USA). The supernatant collected after centrifugation was the exosome-free culture medium.

NK-92 and NK-92MI cell lines were cultured in the above-mentioned exosome-free culture medium (culture medium without EVs) for 2-3 days, and then the following three experimental operations were performed to obtain extracellular vesicles LEV (Large Extracellular Vesicle), extracellular vesicles EXO (Exosome) and extracellular vesicles SEV (Small Extracellular Vesicle).

1.1.1 Isolation and acquisition of extracellular vesicles LEV: ① Collect the culture fluid of the two cell lines after culturing for 2-3 days, centrifuge at 400g for 10 minutes at 4°C, and collect the supernatant; ② Centrifuge the supernatant at 2000g for 20 minutes at 4°C, discard the precipitate at the bottom (the precipitate is cells and debris), and collect the supernatant; ③ Centrifuge the obtained supernatant at 10000g for 30 minutes at 4°C, and then wash the precipitate obtained by centrifugation with phosphate buffered saline (PBS) at 10000g for 30 minutes at 4°C. The precipitates obtained at last are LEVs derived from the two immune cell lines, namely NK-92LEV and NK-92MILEV (as shown in Figure 2; Figure 3); ④ Gently blow, dissolve and mix the LEV precipitates with 50-100μL PBS, respectively, and store them at -80°C. 1.1.2 Isolation and acquisition of extracellular vesicles EXO: ① The supernatant obtained by centrifugation at 10000g for 30min at 4°C in the experimental operation ③ of 1.1.1 was filtered through a sterile filter with a diameter of 0.22μm, and the filtered liquid was collected respectively; ② The filtered collected liquid was centrifuged at 110000g for 70min at 4°C (ultracentrifuge rotor: SW32Ti), and the obtained precipitates were collected respectively; ③ The obtained precipitates were re-washed and suspended with PBS, centrifuged and washed at 110000g for 70min at 4°C, and the PBS resuspension and washing experimental operation was repeated 1-2 times, and finally the precipitates obtained were EXOs derived from two immune cell lines, namely NK-92EXO and NK-92MIEXO (as shown in Figure 2; Figure 3); ④ The EXO precipitates were gently blown, dissolved and mixed with 50-100μL PBS, and stored at -80°C.

1.1.3 Isolation and acquisition of extracellular vesicles SEV: ① The liquid collected after filtration in 1.1.2. Experimental operation ① was centrifuged at a centrifugal force of 110,000 g for 70 min at 4°C, and the supernatant was collected while the precipitate was collected in 1.1.2. Experimental operation ② for separation and acquisition of extracellular vesicles EXO; ② The obtained supernatants were centrifuged at a speed of 167,000 g for 16 h at 4°C, and the obtained precipitates were collected; ③ The obtained precipitates were re-washed and suspended with PBS, centrifuged and washed at a centrifugal force of 167,000 g for 4 h at 4°C, and the PBS resuspension and washing experimental operations were repeated 2-3 times, and the final precipitates obtained were SEVs derived from two immune cell lines, namely NK-92SEV and NK-92MISEV (as shown in Figure 2; Figure 3); ④ The EXO precipitates were gently blown, dissolved and mixed with 50-100 μL PBS, and stored at -80°C.

The process of isolating and obtaining the above-mentioned different types of extracellular vesicles is summarized in FIG1 , and the transmission electron microscopy and particle size characterization of the obtained extracellular vesicles are shown in FIG2 and FIG3 , respectively.

After isolating and obtaining two groups of 6 types of extracellular vesicles using the above experimental method, the RNA adsorbed on the surface of the vesicles was further removed by the following experimental process: the various EVs obtained were resuspended in PBS, and an appropriate amount of RNase (RNaseA, Ambion, TX, USA) was added to make the final concentration of RNaseA 1U/mL, and then the various extracellular vesicle solutions containing RNaseA were incubated in a 37°C water bath (Jing Hong, SHH, CN) for 20 minutes. This experimental procedure follows the guidelines of the International Society for Extracellular Vesicles (ISEV). 1.2 miRNA sequencing and bioinformatics analysis of human cell lines NK-92 and NK-92MI and their secreted extracellular vesicles

1.2.1 miRNA sequencing of NK-92 and NK-92MI cell lines

We used a total RNA purification kit (Norgen Biotek Corp, Thorold, ON, Canada) to extract total RNA (including all miRNAs and small molecule RNAs) from NK-92 and NK-92MI cultured cell samples, respectively. We used Bioanalyzer 2100 (Agilent, CA, USA) to analyze the quality and quantity of the obtained RNA and ensure that the RIN value was >7.0. We took 1 μg of total RNA from the RNA samples extracted from the two cell lines and prepared small molecule RNA libraries using the TruSeq small molecule RNA sample preparation kit (Illumina, SD, USA). According to the operating instructions provided by the manufacturer of Illumina HiSeq 2500 (Hanyu, SHH, China), we commissioned Lianchuan Biotechnology Co., Ltd. (LC Sciences, HZ, CN) to perform 50 bp single-end sequencing of miRNAs in the above two cell line samples.

1.2.2 miRNA sequencing of two groups of extracellular vesicles

In order to remove the RNase added in 1.1 and purify the extracellular vesicles simultaneously, we re-performed ultracentrifugation and washing according to the steps of obtaining and purifying the corresponding extracellular vesicles in 1.1; the total RNA of various extracellular vesicles was extracted and the small molecule RNA library was prepared using the same method as in 1.2.1, and the miRNA in various extracellular vesicle samples was sequenced at 50bp single end.

1.2.3 Bioinformatics analysis of miRNA sequencing results

First, the ACGT101-miR analysis software (LCSciences, TX, USA) was used to perform bioinformatics analysis on the miRNA sequencing data of human cell lines and their secreted extracellular vesicles. The main analysis process of the software is as follows: the raw data is processed by quality control to obtain clean reads, the clean reads are removed from the 3' linker and length-screened, and the sequences with base lengths of 18-26 nt are retained; then the remaining sequences are compared with RNA database sequences, such as the mRNA database, RFam database (including rRNA, tRNA, snRNA, snoRNA, etc.) and Repbase database (repetitive sequence database), and filtered.

Secondly, based on sequencing data and bioinformatics analysis software, miRNAs with potential important biological functions were screened. The main screening methods and processes: ① Based on DESeq2 (V1.26.0) (R language software package), differential expression analysis was performed on the miRNA sequencing results of the two cell lines and the miRNA sequencing results of the two groups of extracellular vesicles. The miRNAs that met the log ₂ FoldChange>1.25 and the statistical analysis p value <0.05 were used as differentially expressed miRNAs and clustered analysis was performed. 15 miRNAs with high expression levels and significant differences (NK92 was significantly downregulated compared with NK92-MI) were screened from the sequencing data of the two cell lines (Figure 5 and Table 1), and 50 miRNAs with high common expression levels were screened from the sequencing data of extracellular vesicles (Figure 7 and Table 2); ② Based on the assumption that functional miRNAs should have advantages in expression levels, the average expression of miRNAs in cell lines and extracellular vesicles was calculated. The expression levels were calculated respectively, and the above differentially expressed miRNAs were ranked based on the average expression levels; ③ According to the expression levels and change folds of the miRNAs screened out in ①, 4 target miRNAs with significantly higher expression in NK-92MI than in NK-92 were finally selected from the miRNAs initially screened out from the above 15 cell lines (we first found through experimental studies that the anti-oral tumor effect of NK-92MI cell line is better than that of NK-92 cell line, the results are shown in Figure 4, and analyzed by One-way ANOVA using GraphPad Prism software; ^* , P<0.05, ^** , P<0.01), including 1 unknown miRNA (bta-miR-2478_L-2) (the difference fold is the most obvious) and 3 known miRNAs (hsa-miR-1260a, hsa-miR-197-3p, hsa-miR-296-5p) (Figure 5). At the same time, two target miRNAs with significantly higher expression in EXO than LEV and SEV were selected from the miRNAs initially screened from the above 50 extracellular vesicle miRNAs (we were the first to find through experimental studies that EXO had significantly better anti-oral tumor effect than LEV and SEV, the results are shown in Figure 6, analyzed by One-way ANOVA using GraphPad Prism software; ^* , P<0.05, ^** , P<0.01, ^*** , P<0.001), including the known hsa-miR-339-5p and hsa-miR-223-3p (Figure 7).

The above 6 target miRNAs are the active miRNAs described in this application, and are labeled as miRNA-X.

Table 1 Sequencing results of the top 15 miRNAs with significant expression changes between NK-92/NK-92MI cell lines

Table 2 Sequencing results of 50 miRNAs with high common expression in EVs of NK-92/NK-92MI

Example 2. Construction of active miRNA-X expression vector

2.1 Construction of active miRNA-X expression vector alone

In this example, Lipofectamine 3000 liposomes were used to construct the expression vector, and the concentration of miRNA-Xmimic and mimic control was set to 60 nM. The experiment was carried out in a 96-well plate, and the total system in each well was 200 μL. The specific operation is as follows:

① Take 0.6 μL miRNA-Xmimic and an equal amount of mimicNC (NC is a nematode miRNA sequence) and add them to 9.4 μL Opti-MEM respectively;

② Add 0.3 μL Lipofectamine 3000 to 9.7 μL Opti-MEM and let stand for 5 minutes;

③ Add ① into ②, blow gently and let stand for 15 minutes to obtain Lipofectamine3000 liposomes expressing miRNA-X and Lipofectamine3000 liposomes expressing mimicNC.

2.2 Construction of expression vector for active miRNA-X

This implementation uses Lipofectamine300 liposomes to construct a combined expression vector, and sets the concentration of miRNA-Xmimic and mimic control to 60nM. The experiment is performed in a 96-well plate, with a total volume of 200μL per well. The specific operation is as follows:

① Take 0.6 μL of miRNA-X1mimics, miRNA-X2mimics and mimicNC and add them to 10 μL of Opti-MEM respectively. Prepare two portions of mimicNC using the same formula and method;

② Prepare two liposome solutions: add 0.6μL Lipofectamine3000 to 20μL opti-MEM and let stand for 5 minutes. Prepare two liposome solutions using the same formula and method;

③ Add miRNA-X1mimics and miRNA-X2mimics solutions prepared in ① to one of the liposome solutions, and add two portions of mimicNC prepared in ① to the other liposome solution; gently blow and let stand for 15 minutes.

The miRNA-X expression vectors and miRNA-X1mimics and miRNA-X2mimics expression vectors constructed in 2.1 and 2.2 above are in fact representative dosage forms for miRNA applications, providing a way of thinking for the subsequent preparation of various drugs based on active miRNA-Xmimics alone or in combination. Subsequently, liposome drugs containing one or more active miRNA-Xmimics and their activators/inhibitors can be prepared, and the administration method is external application or injection.

Example 3. Active miRNA-X transfection efficiency test

In order to verify the transfection efficiency of the six miRNA-X screened in Example 1 in oral tumor cells, we conducted an in vitro transfection experiment. First, the same miRNA-X mimics (RiboLifeScience, JS, CN) as the six active miRNA-X screened in Example 1 were selected and co-incubated with the human oral tumor cell line KB. The specific experimental steps were as follows:

① Inoculation of KB cells: Inoculate KB cells in the logarithmic growth phase into a 6-well plate at 1×10 ⁵ to 5×10 ⁵ cells/well.

② Add the Lipofectamine 3000 liposomes expressing miRNA-X obtained in Example 2.1 into KB cells.

③ After 4 hours of transfection, the medium was changed and KB cells were collected after 48 hours of continuous culture. The transfection efficiency was detected by RT-PCR.

By detecting the expression levels of active miRNA-X in KB oral tumor cells through RT-PCR experiments, we confirmed that the expression levels of the six miRNA-X were significantly increased after transfection of miRNA-Xmimics (Figure 8).

To further verify the transfection efficiency of the six miRNA-X inhibitors screened in Example 1, we co-incubated the corresponding six miRNA-X inhibitors (RiboLifeScience, JS, CN) with the human oral tumor cell line KB. The specific experimental steps were as follows: KB cells in the logarithmic growth phase were inoculated into a 6-well plate at 1×10 ⁵ to 5×10 ⁵ cells/well; the concentrations of miRNA-Xinhibitor and inhibitor control were set to 120 nM. The specific operations were as follows:

①Take 12μL of miRNA-Xinhibitor and inhibitor control and add them into 88μL of opti-MEM respectively;

② Take 7.2μL Lipofectamine 3000 and add it to 92.8μL Opti-MEM. Let it stand for 5 minutes.

③ Then add ① to ②, gently blow and let stand for 15 minutes. Then add it to KB cells. Change the medium 4 hours after transfection, continue to culture for 48 hours, collect KB cells, and use RT-PCR experiment to detect transfection efficiency;

By detecting the expression levels of active miRNA-X in KB oral tumor cells through RT-PCR experiments, we confirmed that the expression levels of the six miRNA-X were significantly decreased after transfection of miRNA-Xinhibitor (Figure 8).

From the above miRNA-Xmimics and inhibitors transfection experiments, we can see that the miRNA-X we screened was successfully transfected into oral tumor cells with the help of the vector system constructed by the above method, and produced expected and ideal regulatory effects on the expression of miRNA-X in tumor cells.

Example 4. Anti-oral tumor activity test of active miRNA-X

To further verify the anti-oral tumor effect of miRNA-X, we co-incubated active miRNA-X mimics/inhibitors with KB human oral tumor cell line. The specific experimental steps are as follows:

The miRNA-Xmimics/inhibitors were transfected into KB human oral tumor cells, and the transfection steps were as described in Example 3; the transfection system was replaced with a solution 4 hours after transfection, and 3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide (MTT, Sigma, DA, DE) was added after continued culture for 48 hours to detect the survival rate of KB cells, which was used to evaluate the anti-tumor effect of miRNA-Xmimics/inhibitors on KB cells.

The experimental results showed that the six miRNA-Xmimics significantly inhibited the proliferation of KB oral tumor cells, while miRNA-Xinhibitors did not inhibit the proliferation of KB tumor cells (Figure 9, analyzed by One-way ANOVA using GraphPadPrism software. ^** , P<0.01; ^*** , P<0.001).

It can be seen that overexpression of any one of the six miRNA-Xs can significantly inhibit the proliferation of oral tumor cells and play an anti-oral tumor role. Those skilled in the art can foresee that the active ingredients developed based on the six miRNA-Xs can also significantly inhibit the proliferation of oral tumor cells and play an anti-oral tumor role, including but not limited to: ① miRNA-X derivatives modified from miRNA-X; ② precursor miRNAs that can be processed into the aforementioned six miRNA-Xs in the host; ③ polynucleotides that can be transcribed by the host to form the precursor miRNAs described in ②; ④ expression vectors containing miRNA-X or the miRNA-X derivatives described in ①, or the expression vectors of the precursor miRNAs described in ②, or the expression vectors of the polynucleotides described in ③ (such as 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 combined therapy

In order to further verify the anti-oral tumor effect of miRNA-X combined therapy, we co-incubated the combined expression vector constructed in 2.2 with the human oral tumor cell line KB. The specific experimental steps are as follows:

① Inoculation of KB cells: Inoculate KB cells in the logarithmic growth phase into a 96-well plate at 1×10 ³ to 5×10 ³ cells/well;

② Add the Lipofectamine 3000 liposomes expressing miRNA-X1mimics and miRNA-X2mimics obtained in Example 2 into KB cells;

③ After continuing to culture for 48 hours, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added to detect the survival rate of KB cells to evaluate the anti-tumor effect of miRNA-Xmimics combined therapy on KB cells.

The experimental results showed that the combination of different miRNA-X can produce a good inhibitory/killing effect on KB cells. For example, there were statistically significant differences in the survival rate of cancer cells between the three miRNA combination groups and the NC control group (Figure 10, analyzed by One-way ANOVA using GraphPad Prism software. *, P < 0.05; **, P < 0.01; ***, P < 0.001). On the other hand, there are differences in the anti-oral cancer effects of different miRNA-X combinations, and specific combinations can more effectively inhibit/kill KB cells. For example, the anti-oral cancer activity of bta-miR-2478-L-2 combined with hsa-miR-197-3p was significantly improved compared with the use of bta-miR-2478-L-2 or hsa-miR-197-3p alone: it increased by 100.98% (P<0.001) compared with bta-miR-2478-L-2 alone, and increased by 38.12% (P<0.01) compared with hsa-miR-197-3p alone.

Different from the combined effect of bta-miR-2478-L-2 and hsa-miR-197-3p, the anticancer activity of bta-miR-2478-L-2 combined with hsa-miR-339-5p was higher than that of hsa-miR-339-5p alone (P<0.05), but similar to that of bta-miR-2478-L-2 alone; the anticancer activity of hsa-miR-339-5p combined with hsa-miR-223-3p was higher than that of hsa-miR-339-5p alone (P<0.05), but similar to that of hsa-miR-223-3p alone.

The degree of enhancement or weakening of the above anticancer activity was calculated by the following formula: (average anticancer effect of the single-use group - average anticancer effect of the combined-use group) / average anticancer effect of the single-use group × 100%.

Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

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.2 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.