CN113230267A

CN113230267A - Application of microRNA sequence in preparation of malignant melanoma gene therapy drug

Info

Publication number: CN113230267A
Application number: CN202110304252.3A
Authority: CN
Inventors: 刘杰
Original assignee: West China Hospital of Sichuan University
Current assignee: West China Hospital of Sichuan University
Priority date: 2021-03-22
Filing date: 2021-03-22
Publication date: 2021-08-10

Abstract

The invention provides a medicament, which contains at least one of miR-34a-5p, miR-182-5p and miR-184-3p in the following gene sequences and/or complementary sequences thereof, and also provides application of the gene sequences and/or complementary sequences thereof in preparing medicaments for treating malignant melanoma. The gene sequences and/or complementary sequences of miR-34a-5p, miR-182-5p and miR-184-3p can effectively inhibit the proliferation of malignant melanoma cells, and a new choice is provided for clinical medication of malignant melanoma gene therapy.

Description

Application of microRNA sequence in preparation of malignant melanoma gene therapy drug

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to application of a microRNA sequence in preparation of a malignant melanoma gene therapy medicine.

Background

Malignant melanoma is a highly malignant tumor that originates in melanocytes, and is frequently found in the skin, mucous membranes and internal organs. In recent years, the incidence of malignant melanoma has been on the rise year by year. The annual incidence rate in the European and American population is 22.7/10 ten thousand, which is about 5.6% of all malignant tumors. Although the incidence of malignant melanoma in China is obviously lower than that of European and American people, the initial diagnosis period of melanoma patients in China is later and the prognosis is worse.

Surgical resection is an important local treatment for patients with early malignant melanoma. The local radiotherapy can be used for the postoperative adjuvant therapy of partial patients and also can be used for the palliative therapy of non-operable patients, such as the treatment of symptomatic brain metastasis, bone metastasis, tumor bleeding and the like, so as to improve symptoms, improve life treatment and prolong the survival time. However, malignant melanoma radiation therapy is relatively insensitive and some patients have difficulty benefiting from radiation therapy. In addition, small molecule targeted therapeutic drugs and immunotherapy can be used for the treatment of malignant melanoma, but still face the difficulty of drug resistance.

Gene therapy is the transfer of exogenous genetic material into target cells by a variety of methods to treat a particular disease. Gene therapy has the possibility of curing some monogenic genetic diseases once or becomes an important means for treating human diseases in the future. Target cells, nucleic acids and transfer methods are three components of gene therapy, where nucleic acid sequences are the key.

microRNA (miRNA) is non-coding RNA with the length of about 18-25 nucleotides, has high conservative property in evolution and good stability in blood, and becomes a research hotspot of biological standards. At present, the expression levels of miR-34a-5p, miR-182-5p and miR-184-3p are related to melanoma, but no report related to the application of the gene sequences of miR-34a-5p, miR-182-5p and miR-184-3p in gene therapy for treating malignant melanoma is found.

Disclosure of Invention

The invention aims to provide a new application of microRNA in a malignant melanoma gene therapy medicine and a malignant melanoma gene therapy medicine.

The invention provides an application of a sequence of any one or more of the following miRNAs and/or a complementary sequence thereof in preparing a medicament for treating malignant melanoma: miR-34a-5p, miR-182-5p and miR-184-3 p.

Further, the above-mentioned drugs are drugs for inhibiting the proliferation of malignant melanoma cells.

The invention also provides a gene therapy medicine for malignant melanoma, which contains the sequence of any one or more of the following miRNAs and/or the complementary sequence thereof: miR-34a-5p, miR-182-5p and miR-184-3 p.

Furthermore, the medicine contains a carrier of the miRNA sequence and/or the complementary sequence thereof.

Further, the vector is a viral vector, preferably a lentivirus, adenovirus, adeno-associated virus, herpes simplex virus or a virus constructed with the stem-loop sequence of the miRNA.

Further, the virus is a virus targeting malignant melanoma, and preferably a virus targeting hTERT and HIF-1 α.

Further, the above-mentioned medicament contains a reagent for transfecting the sequence of the miRNA and/or its complementary sequence.

Further, the agent is a cationic lipid substance or an amphoteric molecular lipid substance, and the amphoteric molecular lipid substance is preferably a liposome.

Experimental results show that the miR-34a-5p, miR-182-5p and miR-184-3p gene sequences and/or complementary sequences thereof are transfected into malignant melanoma cells, so that the proliferation of the malignant melanoma cells can be effectively inhibited.

The hTERT refers to Human telomerase reverse transcriptase (Human telomerase reverse transcriptase);

HIF-1 α refers to hypoxia-inducible factor-1 α (hypoxia-inducer factors-1 α);

lentivirus refers to Lentivirus (LV); adenovirus refers to Adenoviruses (ADV), adeno-associated virus refers to adeno-associated virus (AAV), and Herpes Simplex Virus (HSV);

miR-34a-5p refers to: gene ID 407040; ensembl: ENSG00000284357 MIM: 611172; miRBase MI0000268

miR-182-5p means: gene ID 406958; ensembl: ENSG00000207990 MIM: 611607; miRBase MI0000272

miR-184-3p refers to: gene ID 406960; ensembl: ENSG00000207695 MIM: 613146; MiRBase MI0000481

The "sequence of miRNA and/or its complementary sequence described in the present invention means": the sequence of miRNA, or the complementary sequence of miRNA sequence, or the double strand formed by miRNA sequence and its complementary sequence.

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

FIG. 1 shows the effect of miR-31-5p, miR-34a-5p, miR-129-5p, miR-146a-5p, miR-182-5p and miR-184-3p sequence transfection on tumor cells.

Detailed Description

The raw materials and equipment used in the invention are known products and are obtained by purchasing commercial products.

Example 1 Gene therapy medicine for malignant melanoma

1. Constructing gene sequences of miR-34a-5p, miR-182-5p and miR-184-3p and/or complementary sequences thereof.

2. Construction of vectors for miRNA gene therapy

And constructing a virus vector for miRNA gene therapy by using the Stem-loop sequence of the miRNA.

3. Combining the gene sequence with the carrier to obtain the gene therapeutic medicine for malignant melanoma.

Example 2 Gene therapy medicine for malignant melanoma

Steps 1 to 2 are the same as in example 1.

3. Combining the gene sequence with the virus vector and the liposome transfection reagent to obtain the gene therapy medicine for malignant melanoma.

Example 3 Gene therapy medicine for malignant melanoma

Steps 1 to 2 are the same as in example 1.

3. Combining the gene sequence with liposome transfection reagent to obtain the gene therapeutic medicine for malignant melanoma.

Example 4 Gene therapy for malignant melanoma

1. Constructing gene sequences and/or complementary sequences of miR-34a-5p, miR-182-5p and miR-184-3 p.

2. Expression vector using lentivirus, adenovirus, adeno-associated virus or herpes simplex virus as gene sequence and/or gene complementary sequence

3. Construction of viral vector for targeting miRNA gene therapy of malignant melanoma

Malignant melanoma highly expresses Human telomerase reverse transcriptase (hTERT), while normal Human cells do not normally express or underexpress hTERT. Meanwhile, malignant melanoma often forms hypoxic regions in the process of rapid growth, so that tumor cells highly express hypoxia-inducible factors-1 alpha (HIF-1 alpha). Therefore, the dual specificity promoter regions of hTERT and HIF-1 alpha gene can be used to initiate replication of viruses such as lentivirus, adenovirus, adeno-associated virus, herpes simplex virus, etc.

The characteristic that only tumor cells simultaneously highly express hTERT and HIF-1 alpha is utilized to construct a virus vector with malignant tumor targeting.

4. Combining the gene sequence with the targeted virus vector to obtain the gene therapy medicine for malignant melanoma. The virus is replicated in malignant melanoma cells and expresses miR-34a-5p, miR-182-5p and miR-184-3p, and virus particles released after the death of tumor cells can infect and destroy other tumor cells, so that the miRNA-treated oncolytic virus targeting malignant melanoma is obtained.

The beneficial effects of the present invention are demonstrated by the following experimental examples.

Experimental example 1 Effect of miRNA on tumor cell proliferation

1. Chemically synthesizing miRNA mimics: according to mature sequences (Table 1) of 6 candidate miRNAs (miR-31-5p, miR-34a-5p, miR-129-5p, miR-146a-5p, miR-182-5p and miR-184-3p), miRNA mimics (the sense sequence of the miRNA mimics is completely identical to the mature sequence of the miRNA, and the antisense sequence is completely complementary to the mature sequence of the miRNA) is synthesized by chemotherapy, and the base sequence of the miRNA mimics is shown in Table 2. Suitable Negative Controls (NC) were selected based on the number of bases of the miRNA, wherein the negative control for miR-182-5p is 24 bases (NC-24), the negative controls for the remaining miRNAs are 22 bases (NC-22), and commercially available commercial reagents were used as the negative controls.

TABLE 1 mature sequences of miRNA

TABLE 2 sequence of miRNA mimics

2. Cell culture: malignant melanoma B16F10 cells were added to 96-well plates and cultured using complete medium without antibiotics. The number of cells was 2,500 cells/well and the volume of the medium is shown in Table 3. Culturing for 12h to make the cells grow adherently.

3. Transfection:

(1) transfection reagent: riboFECT using Boehringer creature^TMCP transfection reagents.

(2) Cell transfection: using riboFECT^TMCP Buffer diluted the negative control and candidate miRNA mix (stock solution concentration 20. mu.M), and mixed gently. Preparing a mixed solution: according to the following steps: volume ratio of 10 in riboFECT^TMAdding into CP BufferriboFECT^TMAnd (5) CP Reagent, lightly blowing, beating and uniformly mixing, and incubating at room temperature for 0-15 min. Will riboFECT^TMThe CP mixture was added to the culture medium of B16F10 cells and gently mixed. The specific ratios of the components are shown in Table 3 (refer to riboFECT for details)^TMInstructions for CP transfection reagents). The culture was continued for 60 h.

4. The proliferation of B16F10 cells was examined by CCK-8.

The result is shown in figure 1, and miR-34a-5p, miR-182-5p and miR-184-3p mimics (mimics sequence is base complementary double-stranded RNA containing sense sequence and antisense sequence at the same time) can obviously inhibit the proliferation of B16F10 cells. The proliferation inhibition effect of the miR-184-3p mimics on B16F10 cells is remarkable at the concentrations of 100nM and 200nM, the proliferation inhibition effect of the miR-182-5p mimics on B16F10 cells is remarkable at the concentrations of 100nM and 400nM, and the proliferation inhibition effect of the miR-34a-5p mimics on B16F10 cells is remarkable at the concentration of 400 nM.

miR-146a-5p transfection can promote proliferation of B16F10 cells, and the proliferation of the B16F10 cells is not obviously influenced by the rest candidate miRNAs.

The experimental results show that the miR-34a-5p, miR-182-5p and miR-184-3p sequences and complementary sequences thereof can obviously inhibit the proliferation of malignant melanoma cells. Thus, the compound has important application value in preparing gene therapy medicines for treating malignant melanoma.

TABLE 3 proportion of each component in miRNA mimics transfection procedure

In conclusion, the invention provides application of miR-34a-5p, miR-182-5p and miR-184-3p gene sequences or complementary sequences thereof in preparation of a medicine for treating malignant melanoma, and a medicine for treating malignant melanoma gene prepared by using miR-34a-5p, miR-182-5p and miR-184-3 p. The miR-34a-5p, miR-182-5p and miR-184-3p gene sequences and/or complementary sequences thereof can effectively inhibit the proliferation of malignant melanoma cells, and provide a new choice for clinical medication of malignant melanoma gene therapy.

Claims

1. Use of a sequence of any one or more of the following mirnas and/or a complement thereof in the manufacture of a medicament for the treatment of malignant melanoma: miR-34a-5p, miR-182-5p and miR-184-3 p.

2. The use of claim 1, wherein the medicament is a medicament for inhibiting the proliferation of malignant melanoma cells.

3. A gene therapy medicine for malignant melanoma is characterized by comprising the sequence of one or more miRNA (micro ribonucleic acid) and/or the complementary sequence thereof: miR-34a-5p, miR-182-5p and miR-184-3 p.

4. A medicament as claimed in claim 3, comprising a vector for the miRNA sequence and/or its complement.

5. The medicament of claim 4, wherein the vector is a viral vector.

6. The pharmaceutical of claim 5, wherein the viral vector is a lentivirus, adenovirus, adeno-associated virus, herpes simplex virus, or a virus constructed with the stem-loop sequence of the miRNA.

7. The medicament according to claim 6, wherein the virus is a virus targeting malignant melanoma, preferably a virus targeting hTERT and HIF-1 α.

8. A medicament as claimed in any one of claims 3 to 7, comprising an agent for transfecting the sequence of the miRNA and/or its complement.

9. Pharmaceutical according to claim 8, wherein the agent is a cationic lipid substance or an amphiphilic lipid substance, preferably a liposome.

10. The agent according to any one of claims 3 to 9, which is an agent for inhibiting the proliferation of malignant melanoma cells.