CN102643811B - The antisense oligonucleotide of people miR-1229 and application thereof - Google Patents

Abstract

The invention discloses an antisense oligonucleotide for inhibiting the expression of human microRNA-1229 and its application. The antisense oligonucleotide specifically binds to human miR-1229, and comprises a sequence complementary to 13 to 23 consecutive nucleotides in the following nucleotide sequence: 5'-CUCUCACCACUGCCCUCCCACAG-3', especially comprising Sequence: 5'-CUGUGGGAGGGCAGUGGUGAGAG-3'. The antisense oligonucleotide of the present invention can be ribonucleotide, deoxyribonucleotide or chimera of ribonucleotide and deoxyribonucleotide, and any nucleotide in the chain can be modified. The miR-1229 antisense oligonucleotide of the present invention can effectively inhibit the expression of miR-1229 in human brain glioma cells, inhibit its growth and proliferation, thereby effectively treating brain glioma and other tumors with high miR-1229 expression .

Description

Antisense oligonucleotide of human miR-1229 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 microRNAs (miRNA) antisense oligonucleotide, in particular to human microRNA-1229 (human miR-1229) antisense oligonucleotide and applications thereof. The antisense oligonucleotide can be complementary to human miR-1229, thereby inhibiting the expression of human miR-1229 to play an anti-tumor effect. The present invention also relates to a pharmaceutical composition comprising the miRNA antisense oligonucleotide.

Background technique

miRNAs (also known as microRNA or microRNA) are small non-coding RNAs with a length of 20-25bp, usually transcribed by RNA polymerase II (PolII), and generally the initial product is a large cap structure (7MGpppG) and multiple pri-miRNA (primarymiRNA, primary miRNA) of polyadenylic acid tail (AAAAA). These pri-miRNAs are processed into 70 nucleotide precursor miRNAs (precursormiRNA, pre-miRNA) under the action of RNaseIIIDrosha and its cofactor Pasha. RAN-GTP and exportin5 (exportin 5) transport this precursor molecule into the cytoplasm. Subsequently, another RNaseIII Dicer (endoribonuclease) cuts it to produce a double strand of about 22 nucleotides in length. This double strand is quickly guided into the miRISC (miRNA-induced silencing complex, miRNA-induced silencing complex) complex, which contains Argonaute protein (AGO protein), and mature single-stranded miRNA remains 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 post-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 (FlanaganWM. Antisensecomesofage. 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-1229 (hsa-miR-1229) is located on chromosome 5, the precursor sequence is GUGGGUAGGGUUUGGGGGAGAGCGUGGGCUGGGGUUCAGGGACACCCCUCUCACCACUGCCCUCCCACAG (SEQ ID No.1), and contains a mature microRNA: hsa-miR-1229 (MIMAT0005UCACQ.No.1) No. GCCCACC . Dudziec et al. used microarray technology to find that the expression of some miRNAs was up-regulated after 5-azacytidine (5-azacytidine nucleoside) treatment in urothelial cell carcinoma, suggesting that some miRNAs were affected by methylation. Studies have shown that the promoter region of miR-1229 is highly methylated, and the expression of miR-1229 is also closely related to the diagnosis of urothelial cell carcinoma (Dudziec et al., 2010). However, there is no research report on the function and expression level of miR-145 in glioma.

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

Aiming at the deficiencies in the prior art, the present invention designs a series of antisense oligonucleotide molecules that can bind to different positions of miR-1229, and verifies the ability to specifically inhibit the expression of miR-1229 in cultured cells U87/MG The effect of antisense oligonucleotides on cell growth and proliferation. Antisense oligonucleotides can contain 13 to 23 nucleotide residues in length, and they all have varying degrees of inhibition of human tumor cell growth, The characteristics of proliferation ability, wherein the shortest antisense oligonucleotide length is 13 bases, and antisense oligonucleotides of different lengths all have good tumor cell growth and proliferation inhibitory activities. Therefore, the above-mentioned antisense oligonucleotides can all be used to prepare preparations for inhibiting the growth and proliferation of tumor cells, among which tumor cells with high expression of miR-1229 are preferred. The present invention has been accomplished on this basis.

Therefore, the main problem to be solved by the present invention is to provide a group of new antisense oligonucleotides (inhibitors) of human miR-1229, which are used to inhibit the expression of miR-1229 efficiently, with low toxicity or non-toxicity, and then Treatment of diseases caused by overexpression of miR-1229, including tumors, especially glioma.

Another problem to be solved by the present invention is to provide the use of the above-mentioned antisense oligonucleotide in the preparation of medicines for treating diseases related to overexpression of mir-1229.

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

Through extensive and in-depth research, the present inventors designed and synthesized a series of antisense nucleic acids (antisense oligonucleotides) with different lengths specific to different regions of miR-1229, and verified that they have inhibitory effect in cultured cells. Effect of antisense nucleic acid. Studies have shown that these antisense nucleic acids can inhibit the growth and malignant proliferation of tumor cells. The first aspect of the present invention provides an antisense oligonucleotide of human miR-1229, said antisense oligonucleotide inhibits the expression of miR-1229 in human cells. Usually, the antisense oligonucleotide is complementary to 13-23 consecutive nucleotide sequences in 5'-CUCUCACCACUGCCCUCCCACAG-3'. Usually the length of the antisense oligonucleotide is 13-35 bp, and for miRNA mature body, the preferred length of the antisense oligonucleotide is 18-22 bp. 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. In a preferred embodiment of the present invention, the length of the antisense oligonucleotide is 18-23 nucleotides. More preferably, the sequence of the antisense oligonucleotide is 5'-CUGUGGGAGGGCAGUGGUGAGAG-3' (SEQ ID No. 3).

In another preferred embodiment of the present invention, the nucleotides in the antisense oligonucleotides of the present invention can be ribonucleotides, deoxyribonucleotides or ribonucleotides and deoxyribonucleosides acid chimera. 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-1229.

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-1229. In the antisense nucleic acid research of the present invention, there are many various chemical modification methods, including one or more combinations selected from 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 used, such as one or more selected from cholesterol modification, PEG modification, thio modification and 2'-methoxy modification. species etc. The antisense oligonucleotides described herein are modified with 2'-methoxy substitutions.

The antisense nucleic acid of the present invention has the effect of inhibiting the expression of miR-1229. When the above antisense nucleic acid is transfected into the cell line U87 with high expression of miR-1229, 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, there is provided the use of the antisense oligonucleotide of the present invention in the preparation of drugs for the treatment of the following diseases, wherein the antisense oligonucleotide is administered in combination with other therapeutic drugs ; The disease is a disease related to the overexpression of human miR-1229, 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. Because antisense nucleotides can only combine with reverse complementary target sequences, they have the characteristics of high specificity and few side effects.

The antisense oligonucleotide of human miR-1229 provided by the present invention can complement human miR-1229, thereby inhibiting the expression of human miR-1229 and playing an anti-tumor effect, which has the following advantages:

1. The antisense oligonucleotides provided by the present invention 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 85%.

Description of drawings

Figure 1 shows that miR-1229 antisense oligonucleotides inhibit the growth and proliferation of tumor cell U87/MG cells, wherein Figure 1A is a micrograph of U87/MG cells transfected with a FAM-labeled negative control (20 ×); Figure 1B is a micrograph of U87/MG cells transfected with a FAM-labeled negative control (20×, under fluorescence). Figure 1C is a U87/MG cell transfected with a negative control (5'-CAGUACUUUUGUGUAGUACAA-3') Micrograph of cell state (10×); Figure 1D is a micrograph of U87/MG cell state after transfection of miR-1229 antisense oligonucleotide (5'-CUGUGGGAGGGCAGUGGUGAGAG-3') (10×).

Detailed ways

The sequence of the antisense oligonucleotide of the present invention is complementary to 13-23 consecutive nucleotide sequences in 5'-CUCUCACCACUGCCCUCCCACAG-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'-CUGUGGGAGGGCAGUGGUGAGAG-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'-position 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 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: Detection of antisense oligonucleotides of human miR-1229 on human glioma cell line U87/MG inhibitory activity

First, the miR-1229 antisense oligonucleotide was synthesized by Shanghai Gemma Pharmaceutical Technology Co., Ltd., the sequence is: 5'-CUGUGGGAGGGCAGUGGUGAGAG-3'. All the sequences used in the examples were synthesized by Shanghai Gemma Pharmaceutical Technology Co., Ltd.

Cell culture:

U87/MG cells (human brain astroglioblastoma, purchased from the Cell Bank of the Type Culture Collection Committee of the Chinese Academy of Sciences) were purchased from Gibco in 10% FBS-DMEM medium (FBS (fetal bovine serum), DMEM (a A regular medium) purchased from Hyclone) and cultured at 37°C, 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 oligomer-Lipofectamine ^TM 2000 (a transfection reagent) complex as follows:

a. Use 25 μl serum-free I medium (Gibco) diluted miR-1229 antisense oligonucleotide (5'-CUGUGGGAGGGCAGUGGUGAGAG-3'), negative control (SEQ ID No.4: 5'-CAGUACUUUUGUGUGUAGUACAA-3'), FAM-labeled negative control, respectively, respectively. After adding to the wells, the final concentration is 50nM, mix gently, and set 3 duplicate wells for each transfection;

b. Gently mix Lipofectamine ^TM 2000 (Invitrogen) before use, then take 0.25μl for use Dilute it to 25 μl in medium I, mix gently and incubate at room temperature for 5 minutes;

c. After incubation for 5 minutes, the diluted Lipofectamine ^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 nucleotides and controls was 50 nM.

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 the FAM-labeled negative control (Figure 1A, Figure 1B); The light intensity was strong (Fig. 1C); most U87/MG cells died after transfection with miR-1229 antisense oligonucleotide (Fig. 1D).

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, 100 μl of DMSO was added to each well and the absorbance value of OD570-OD630 was read by a microplate reader (see Table 1).

Table 1

Calculate the inhibition rate:

The calculated inhibition rate of cell growth was 85.03±1.87%.

The results show that: the human miR-1229 antisense oligonucleotide provided by the invention has a good inhibitory effect, and the inhibitory rate on the growth of U87/MG exceeds 85%.

Claims (7)

1. the purposes of the antisense oligonucleotide of a people microRNA-1229 in the medicine of preparation treatment cerebral glioma, it is characterized in that, described antisense oligonucleotide is made up of the sequence with following nucleotide sequence complementation: 5 '-CUCUCACCACUGCCCUCCCACAG-3 '.

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

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

4. the purposes according to any one of claims 1 to 3, is characterized in that, described antisense oligonucleotide is modified further.

5. purposes as claimed in claim 4, is characterized in that, described modification is selected from one or more in ribose modification, base modification and phosphate backbones modification.

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

7. purposes as claimed in claim 1, is characterized in that, described antisense oligonucleotide and other treatment is medication combined uses.