CN102140466A - Human miR-1825 antisense nucleic acid and application thereof - Google Patents

Abstract

The invention discloses an antisense oligonucleotide for inhibiting the expression of human microRNA-1825 and its application. The antisense oligonucleotide specifically binds to human miR-1825, and contains a sequence complementary to at least 13 consecutive nucleotides in the 5'-UCCAGUGCCCUCCUCUCC-3' nucleotide sequence, especially the sequence: 5'-GGAGAGGAGGGCACUGGA -3'. The antisense oligonucleotide of the present invention can be ribonucleotide, deoxyribonucleotide or a chimera of the two, and any nucleotide in the chain can be modified. The miR-1825 antisense oligonucleotide of the present invention can effectively inhibit the expression of miR-1825 in human glioma cells, inhibit the growth and proliferation of the cells, thereby effectively treating glioma and other tumors with high expression of miR-1825 .

Description

Human miR-1825 antisense nucleic acid and its application

technical field

The invention belongs to the technical field of biomedical materials and the field of medicine. Specifically, the present invention relates to a use of microRNAs (miRNA), in particular to human microRNA-1825 (human miR-1825) antisense nucleic acid and its application. The antisense nucleic acid can be complementary to human miR-1825, thereby inhibiting the expression of human miR-1825 to play an anti-tumor effect. The invention also relates to a pharmaceutical composition containing the miRNA antisense nucleic acid.

Background technique

miRNAs are small non-coding RNAs with a length of 20-25bp, usually transcribed by RNA polymerase II (Pol II), and generally the initial product is a large one with a cap structure (7MGpppG) and a polyadenylic acid tail (AAAAA) The pri-miRNA. These pri-miRNAs are processed into pre-miRNA precursor products consisting of 70 nucleotides under the action of RNase III Drosha and its cofactor Pasha. RAN-GTP and exportin 5 transport this precursor molecule into the cytoplasm. Subsequently, another RNase III Dicer cuts it to produce a double strand of about 22 nucleotides in length. This double strand is quickly guided into the (miRISC) complex, which contains the Argonaute protein, and the mature single-stranded miRNA is retained in this complex. Mature miRNAs bind to the sites of their complementary mRNAs to negatively regulate gene expression through two mechanisms that depend on sequence complementarity, and miRNAs that are not fully complementary to target mRNAs repress their expression at the level of protein translation. However, recent evidence also suggests that these miRNAs may also affect mRNA stability. The miRNA binding site using this mechanism is usually in the 3' untranslated region of the mRNA. If miRNAs are perfectly complementary (or almost perfectly complementary) to the target site, binding of these miRNAs tends to cause degradation of the target molecule's mRNA. miRNAs are quite conservative in the evolution of species, and the expression of miRNAs found in animals, plants and fungi has strict tissue specificity and timing.

Currently, only a small fraction of the biological functions of miRNAs have been elucidated. These miRNAs regulate cell growth and tissue differentiation, and are related to biological growth and development. A series of studies have shown that miRNAs play an important role in cell growth and apoptosis, blood cell differentiation, homeobox gene regulation, neuronal polarity, insulin secretion, brain morphogenesis, cardiogenesis, and later embryonic development. For example, miR-273 is involved in the development of the nervous system of nematodes; miR-430 is involved in the brain development of zebrafish; miR-181 controls the differentiation of mammalian hematopoietic cells into B cells; miR-375 regulates the development of mammalian islet cells and insulin secretion; -143 plays a role in adipocyte differentiation; miR-196 is involved in the formation of mammalian limbs, and miR-1 is related to heart development. Other researchers found that many nervous system miRNAs were temporally regulated in cortical cultures, suggesting that they may control regionalized mRNA translation.

miRNA expression is associated with a variety of cancers, and these genes may function as tumor suppressors or oncogenes. Changes in the expression levels of miRNAs were first found in B-cell chronic lymphocytic leukemia (CLL), and then changes in the expression levels of miRNAs were detected in various human tumors. Studies have found that miRNAs are associated with tumorigenesis, acting as both tumor suppressor genes (such as miR-15a and miR-16-1) and oncogenes (such as miR-155 and miR-17-92 cluster ). At present, it is believed that in tumor cells, some miRNA mature or precursor expression levels are abnormal, and the abnormally expressed miRNA plays a role by affecting the translation of target mRNA, participates in the process of tumor formation, and plays an important role. For example, the Ras proto-oncogene is regulated by the let-7 family, the BCL2 anti-apoptotic gene is regulated by the miR-15a-miR-16-1 cluster, the E2F1 transcription factor is regulated by the miR-17-92 cluster, and the BCL6 anti-apoptotic gene is regulated by the miR -127 regulation and so on. The down-regulation of miRNAs is also closely related to tumorigenesis, which indicates that miRNAs have the function of oncogenes. For example, miR-143 and miR-145 were significantly downregulated in colon cancer. Interestingly, the hairpin precursor molecules were found in similar amounts in tumor and normal tissues, suggesting that the downregulation of miRNAs may be due to disrupted processing. However, the tumor suppressor gene functions of miR-143 and miR-145 may not be limited to colon cancer, and their expression levels are also significantly down-regulated in cell lines such as breast cancer, prostate cancer, uterine cancer, and lymphoma. Another report showed that miR-21 expression was increased in glioblastoma. The expression of this gene in tumor tissue is 5-100 times higher than in normal tissue.

miRNAs are natural antisense factors that can regulate a variety of genes related to the survival and proliferation of eukaryotes. In the aspect of tumor therapy, the application prospect of miRNA is bright. In terms of using miRNA as a therapeutic target, existing experimental data support: for example, during the treatment of gemcitabine (gemcitabine), there are changes in the expression profile of miRNA; regulating the expression level of some miRNA (such as overexpressing miR-21), can Enhance the sensitivity of cholangiocarcinoma cells to chemotherapeutic drugs. By introducing synthetic antisense oligonucleotides complementary to miRNAs with oncogene properties—anti-miRNA oligonucleotides (AMOs)—it is possible to effectively inactivate miRNAs in tumors and delay their growth. Clinically, miRNA can be inactivated by frequent or continuous 2'-O-methylation or the administration of modified antisense oligonucleotides such as locked nucleic acid (LNA). These modifications make the oligonucleotides more stable and less toxic than other treatments. Using antagomirs (Cholesterol-conjugated AMOs), which can effectively inhibit miRNA activity in different organs after injection into mice, may become a promising therapeutic drug. Conversely, overexpression of miRNAs that act as tumor suppressor genes, such as the let-7 family, can also be used to treat certain tumors.

Antisense oligonucleotide (Flanagan WM. Antisense comes of age. Cancer & Metastasis Reviews 1998; 17(2): 169-76) refers to a nucleotide that can be complementary to the base of its target gene. Antisense oligonucleotides can inhibit the expression of corresponding genes.

Human microRNA-1825 (has-miR-1825) is located on chromosome 20, and its precursor sequence is AGAGACUGGGGUGCUGGGCUCCCCUAGACUAGGACUCCAGUGCCCUCCUCUCC. There is one mature microRNA: hsa-miR-1825 (MIMAT0006765, the sequence is UCCAGUGCCCUCCUCUCC). There are no research reports on the function and expression level of microRNA-1825.

In the past 30 years, although the comprehensive treatment of tumors has been very common clinically, the comprehensive treatment based on surgery and supplemented by radiotherapy and chemotherapy has not significantly improved the survival rate of cancer patients, and the 5-year overall survival rate is still low, hovering At about 30% to 55%, there is no significant improvement, and the 5-year survival rate of middle and advanced patients is even lower, about 20%. Moreover, these methods all have their own limitations, especially the curative effect is not good for middle-advanced and relapsed patients, and the curative effect is even worse for those with distant metastasis. Therefore, finding a safer and more effective treatment approach is an urgent problem to be solved to improve the survival rate and quality of life of cancer patients.

Contents of the invention

The main problem to be solved by the present invention is to provide a group of novel antisense nucleic acids (inhibitors) of miR-1825, which are used to inhibit the expression of miR-1825 with high efficiency, low toxicity or non-toxicity, and then treat the miR-1825 overexpression Caused diseases, including tumors, especially gliomas.

Another problem to be solved by the present invention is to provide the use of the above-mentioned antisense oligonucleotides in the preparation of medicines for treating diseases related to the overexpression of mir-1825.

Another problem to be solved by the present invention is to provide a pharmaceutical composition comprising the above-mentioned antisense nucleic acid.

Through extensive and in-depth research, the inventors designed and synthesized a series of antisense nucleic acids with different lengths specific to different regions of miR-1825, and verified the antisense nucleic acids with inhibitory effect in cultured cells. Studies have shown that these antisense nucleic acids can inhibit the growth and malignant proliferation of tumor cells.

The present invention designs a series of antisense nucleic acid molecules that can bind to different positions of miR-1825, and verifies the effect of antisense nucleic acid that specifically inhibits the expression of miR-1825 on cell growth and proliferation in cultured cells U87/MG , the length of antisense nucleic acid molecules can contain 13-25 nucleotide residues, all of which have the characteristics of inhibiting the growth and proliferation of human tumor cells to varying degrees. The shortest antisense nucleic acid is 13 bases in length, and the All the antisense nucleic acids have good tumor cell growth and proliferation inhibitory activities. Therefore, the above-mentioned antisense nucleic acids can be used to prepare preparations for inhibiting the growth and proliferation of tumor cells, among which tumor cells with high expression of miR-1825 are preferred. The present invention has been accomplished on this basis.

The first aspect of the present invention provides an antisense oligonucleotide of miR-1825, said antisense oligonucleotide inhibits the expression of miR-1825 in human cells. Usually, the antisense oligonucleotide is complementary to 13-18 consecutive nucleotide sequences in 5'-UCCAGUGCCCUCCUCUCC-3'. In a preferred embodiment of the present invention, the length of the antisense oligonucleotide is 13-18 nucleotides. More preferably, the sequence of the antisense oligonucleotide is 5'-GGAGAGGAGGGCACUGGA-3'.

From the current point of view, the hybridization affinity of RNA and miRNA in nucleic acid hybridization is higher than that of DNA and miRNA, which has high medicinal value. However, the cost of artificially synthesized DNA is far lower than that of synthetic RNA, and it also has good market potential. Moreover, the antisense nucleic acid formed by the chimeric connection of ribose RNA monomer and deoxyribose DNA monomer can also be used as a drug for development. A series of antisense nucleic acid molecules designed in the present invention include both DNA molecules and RNA molecules, both of which have the activity of inhibiting the expression of miR-1825.

The sequence of the antisense nucleic acid designed in the present invention has specific biological activity, and it has a great relationship with the complementary length of the antisense nucleic acid at the complementary site of a certain gene. If the complementary one is longer, the biological activity will decrease. Higher, the inhibitory effect will be better, and the antisense nucleic acid that is complementary to the same gene site by adding or reducing one to several bases also has different degrees of biological activity, and can also achieve different degrees of antisense nucleic acid. Inhibiting the growth and proliferation of tumor cells, the research of the present invention shows that the shortest 13 bases still have the effect of inhibiting the expression of miR-1825. In antisense nucleic acid research, there are many chemical modification methods, including one or a combination of ribose modification, base modification and phosphate backbone modification. It should be clear that any modification method that can increase the stability and bioavailability of antisense nucleic acid can be applied, such as cholesterol modification, PEG modification, thio modification, 2'-methoxy modification, etc. The antisense oligonucleotides described herein are 2' methoxy modified.

The antisense nucleic acid of the present invention has the effect of inhibiting the expression of miR-1825. When the above antisense nucleic acid is transfected into the cell line U87 with high miR-1825 expression, it can effectively inhibit the growth and malignant proliferation of tumor cells.

The present invention also provides a pharmaceutical composition, which contains a safe and effective amount of the oligonucleotide of the present invention and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of injection, for example, by conventional methods using physiological saline or aqueous solution containing glucose and other adjuvants.

The "effective amount" refers to the amount that can produce functions or activities on humans and/or animals and can be accepted by humans and/or animals.

Said "pharmaceutically acceptable" ingredients are substances suitable for use in humans and/or animals without undue adverse side effects (such as toxicity, irritation and allergic reactions), ie substances with a reasonable benefit/risk ratio.

In the third aspect of the present invention, the use of the antisense oligonucleotide of the present invention is provided for the preparation of drugs for the treatment of the following diseases: treating diseases related to the overexpression of miR-1825 in the human body, including tumors, preferably brain Glioma.

As used herein, "antisense oligonucleotide" refers to an antisense nucleotide oligomer. Antisense oligonucleotides form triple strands (antigene) with double-stranded DNA through complementary base (A-T, A-U, G-C) pairing, or form hybrid double strands (antisense) with single-stranded RNA, thereby blocking gene expression. Replication, transcription, or post-transcriptional processing and translation of mRNA. At the same time, double-stranded RNA can be degraded by intracellular ribonuclease H (RNaseH), thereby more effectively blocking the expression of target genes. Since the antisense nucleotide can only be combined with the reverse complementary target sequence, it has the characteristics of high specificity and small side effects.

The length of the antisense oligonucleotide of the present invention is not particularly limited. Generally speaking, in order to achieve hybridization specificity, the antisense oligonucleotide needs at least 13 nucleotides composed of monomers. Usually the length of the antisense oligonucleotide is 13-35 bp, and for miRNA mature body, the preferred length of the antisense oligonucleotide is 18-25 bp.

Description of drawings

Figure 1 shows that miR-1825 antisense oligonucleotides inhibit the growth and proliferation of tumor cell U87/MG cells, A, B are U87/MG cells transfected with FAM-labeled negative control; C is transfection negative The state of U87/MG cells after control (5'-CAGUACUUUUGUGUAGUACAA-3'); D is the state of U87/MG cells after transfection of miR-1825 antisense oligonucleotide (5'-GGAGAGGAGGGCACUGGA-3').

Detailed ways

The sequence of the antisense oligonucleotide of the present invention is complementary to 13-25 consecutive nucleotide sequences in 5'-UCCAGUGCCCUCCUCUCC-3', and is not complementary to RNA sequences of other genes. In a preferred embodiment of the present invention, the sequence of the antisense oligonucleotide is 5'-GGAGAGGAGGGCACUGGA-3'. The antisense oligonucleotide provided by the present invention can be a modified product, which contains at least two, usually at least 4, preferably at least 6, and more preferably at least 8 nucleotides modified cores without toxic side effects Nucleic acid, the modification method includes 2' methoxy substitution, thio modification, etc. In order to increase the cellular uptake rate of the antisense oligonucleotide, the antisense oligonucleotide can also be modified with cholesterol or PEGylated on the basis of the above modifications. The above-mentioned modified oligonucleotides can continue to effectively pair with the target sequence, and have a longer half-life in vivo than ordinary unmodified ribonucleic acid or deoxyribonucleic acid.

The present invention has the following advantages:

1. Antisense oligonucleotides act on specific target sites, with few non-specific binding sites and high specificity;

2. The antisense oligonucleotide provided by the present invention has the characteristics of low toxicity, small side effects and long half-life through appropriate chemical modification;

3. The antisense oligonucleotide provided by the present invention has a good inhibitory effect, and the inhibitory rate on tumor cell growth exceeds 75%.

The present invention will be described in further detail below in conjunction with the embodiments and accompanying drawings. However, it should be understood that these examples are listed for illustrative purposes only, and are not intended to limit the scope of the present invention.

Example

Example 1. Detection of inhibitory activity of miR-1825 antisense oligonucleotides on human glioma cell line U87/MG

First, miR-1825 antisense oligonucleotide was synthesized by Shanghai Gemma Pharmaceutical Technology Co., Ltd., the sequence is: 5'-GGAGAGGAGGGCACUGGA-3'. All the sequences used in the examples were synthesized by Shanghai Gemma Pharmaceutical Technology Co., Ltd.

Cell culture:

U87/MG cells (purchased from the Cell Bank of the Type Culture Collection Committee of the Chinese Academy of Sciences) were cultured in 10% FBS-DMEM medium (FBS was purchased from Gibco, and DMEM was purchased from Hyclone) at 37°C and 5% CO ₂ . U87/MG cells in good growth state were collected, counted by centrifugation, spread in a 96-well plate at 2×10 ³ per well, and cultured at 37° C., 5% CO ₂ for 24 hours.

Transfection:

1) One day before transfection, inoculate cultured cells in a 96-well plate with an appropriate amount of culture medium without antibiotics, so that the confluence of cells at the time of transfection reaches 30-50%;

2) Transfection samples Prepare the oligomer-Lipofecta mine ^TM 2000 complex as follows:

I培养基(Gibco)分别稀释miR-1825反义寡聚核苷酸(5′-GGAGAGGAGGGCACUGGA-3′)、阴性对照(5’-CAGUACUUUUGUGUAGUACAA-3’)、FAM标记的阴性对照，加入孔内后终浓度为50nM，轻轻混匀，每个转染设3个复孔；a. Use 25μl serum-free Opti-MEM

Dilute miR-1825 antisense oligonucleotide (5′-GGAGAGGAGGGCACUGGA-3′), negative control (5′-CAGUACUUUUGUGUAGUACAA-3′) and FAM-labeled negative control in I culture medium (Gibco), and add them to the wells The final concentration is 50nM, mix gently, and set up 3 replicate wells for each transfection;

I培养基，轻轻混匀后在室温下孵育5min；b. Gently mix Lipofecta mine ^TM 2000 (Invitrogen) before use, then take 0.25 μl of Opti-MEM diluted to 25 μl

I culture medium, mix gently and incubate at room temperature for 5 min;

c. After incubation for 5 minutes, the diluted Lipofecta mine ^TM 2000 was mixed with the diluted antisense nucleotide and the control respectively, mixed gently and then incubated at room temperature for 20 minutes to allow the formation of complexes;

3) The complex was added to each well containing cells and medium, and the culture plate was gently shaken back and forth to mix; the final concentration of antisense nucleotide and control was 75nM.

4) After continuing to incubate for 72 hours in a 5% CO ₂ incubator at 37° C., the U87/MG cells were observed under a microscope and photographed.

As shown in Figure 1, after 72 hours of transfection, more than 80% of U87/MG cells were successfully transfected with FAM-labeled negative control (Figure A, B); after transfection of negative control, U87/MG cells were intact and light-transmissive strong (Panel C); most U87/MG cells died after transfection of miR-1825 antisense oligonucleotides (Panel D).

MTT-based cytotoxicity assay:

Add the prepared MTT (Sigma) 5 mg/ml (prepared with 0.9% physiological saline) to the cells obtained in the previous step, add 20 μl to each well, incubate at 37°C, 5% CO ₂ for 4 hours, then suck out the medium and For MTT, add 100 μl of DMSO to each well and read the absorbance value of OD570-OD630 with a microplate reader.

Calculate the inhibition rate:

The calculated inhibition rate of cell growth was 76.23±2.14%.

The results show that: the miR-1825 antisense oligonucleotide provided by the invention has a good inhibitory effect, and the inhibitory rate on the growth of U87/MG exceeds 75%.

Claims (10)

1. an antisense oligonucleotide is characterized in that, described antisense oligonucleotide comprise with following nucleotide sequence in 13～25 successive Nucleotide complementary sequence: 5 '-UCCAGUGCCCUCCUCUCC-3 '.

2. antisense oligonucleotide as claimed in claim 1 is characterized in that, described antisense oligonucleotide sequence is 5 '-GGAGAGGAGGGCACUGGA-3 '.

3. antisense oligonucleotide as claimed in claim 1 is characterized in that, described antisense oligonucleotide is the mosaic of ribonucleotide, deoxyribonucleotide or ribonucleotide and deoxyribonucleotide.

4. as each described antisense oligonucleotide in the claim 1～3, it is characterized in that described antisense oligonucleotide is further modified.

5. antisense oligonucleotide as claimed in claim 4 is characterized in that, described modification is selected from one or more the combination in ribose modification, base modification and the phosphoric acid backbone modification.

6. antisense oligonucleotide as claimed in claim 5 is characterized in that, described modification is selected from one or more in thio-modification, 2 '-methoxyl group modification and the cholesterol modification.

7. be used to prepare the purposes of medicine of the relative disease of treatment mir-1825 overexpression as each described antisense oligonucleotide in the claim 1～6.

8. purposes as claimed in claim 7 is characterized in that, described antisense oligonucleotide and other treatment are medication combined to be used.

9. purposes as claimed in claim 7 is characterized in that the relative disease of described miR-1825 overexpression is a tumour, is preferably cerebral glioma.

10. a pharmaceutical composition is characterized in that, contains each described antisense oligonucleotide and pharmaceutically acceptable carrier in the claim 1～6 for the treatment of significant quantity.

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