CN102382824A - Human miR-145 antisense nucleic acid and application thereof - Google Patents

Abstract

The invention discloses an antisense oligonucleotide for inhibiting the expression of microRNA-145 and its application. The antisense oligonucleotide specifically binds to human miR-145 and contains a sequence complementary to at least 13 consecutive nucleotides in the 5'-GUCCAGUUUUCCCAGGAAUCCCU-3' nucleotide sequence, especially the sequence: 5'-AGGGAUUCCUGGGAAAACUGGAC -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-145 antisense oligonucleotide of the present invention can effectively inhibit the expression of miR-145 in human brain glioma cells, inhibit its growth and proliferation, thereby effectively treating brain glioma and other tumors with high miR-145 expression.

Description

Human miR-145 antisense nucleic acid and its application

technical field

The invention relates to the field of biomedicine. Specifically, the present invention relates to a use of microRNA (miRNA), in particular to an antisense oligonucleotide for inhibiting the expression of human microRNA-145 (miR-145) and its application. The antisense oligonucleotide can be complementary to human miR-145, thereby inhibiting the expression of human miR-145 to play an anti-tumor effect. The present invention also relates to a pharmaceutical composition containing the miRNA antisense oligonucleotide.

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 RNaseIII 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; 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.

人microRNA-145(hsa-mir-145)位于5号染色体，前体序列为CACCUUGUCCUCACGGUCCAGUUUUCCCAGGAAUCCCUUAGAUGCUAAGAUGGGGAUUCCUGGAAAUACUGUUCUUGAGGUCAUGGUU，含有两个成熟microRNA：hsa-miR-145(序列为GUCCAGUUUUCCCAGGAAUCCCU)和hsa-miR-145*(序列为GGAUUCCUGGAAAUACUGUUCU) . miR-145 was found to be abnormally expressed in human liver cancer cell lines HuH7, HCC, prostate cancer, lung cancer, colon cancer, breast cancer and thyroid cancer (Chang, K.H., P. Mestdagh, et al. 2008; Chiyomaru, T., H .Enokida, et al.2009; Amdt, GM., L.Dossey, et al.2009.), and the characteristic expression of miR-145 is lower in colorectal cancer tissues with tumor diameter greater than 50mm; From normal breast tissue to breast cancer with high proliferation index, the expression of miR-145 also gradually decreased; miR-145 also showed a trend of gradually decreasing expression in HCC tumor histological grades from low to high (Slaby, O ., M. Svoboda, et al. 2007). 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

The main problem to be solved by the present invention is to provide a new antisense oligonucleotide (inhibitor) of miR-145, which is used to inhibit the expression of miR-145 with high efficiency, low toxicity or non-toxicity, and then treat miR-145 Diseases related to overexpression of -145 include various solid tumors, various leukemias, etc.

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-145.

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 inventors designed and synthesized an antisense oligonucleotide specifically targeting miR-145, and verified in cultured cells that it has the effect of inhibiting the expression of miR-145 and inhibiting cell growth. Studies have shown that these antisense nucleic acids can inhibit the growth and malignant proliferation of tumor cells.

The present invention designs an antisense nucleic acid molecule that can specifically bind to miR-145, and verifies the effect of the antisense nucleic acid that specifically inhibits the expression of miR-145 on cell growth and proliferation in cultured cells U87/MG . Antisense nucleic acid molecules can contain 13 to 23 nucleotide residues in length, and all have different degrees of characteristics of inhibiting the growth and proliferation of human tumor cells. The shortest antisense nucleic acid is 13 bases in length. Antisense nucleic acid has good tumor cell growth and proliferation inhibitory activity. 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-145 are preferred. The present invention has been accomplished on this basis.

The first aspect of the present invention provides an antisense oligonucleotide of miR-145, said antisense oligonucleotide inhibits the expression of miR-145 in human cells. Usually, the antisense oligonucleotide is complementary to 13-23 consecutive nucleotide sequences in 5'-GUCCAGUUUUCCCAGGAAUCCCU-3'. 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'-AGGGAUUCCUGGGAAAACUGGAC-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 oligonucleotide molecules designed in the present invention can be DNA, RNA, or a chimera of DNA and RNA. All of the above molecules have the activity of inhibiting the expression of miR-145.

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. Inhibition of tumor cell growth and proliferation. The research of the present invention shows that the shortest 13 bases can still inhibit the expression of miR-145. In the research of antisense nucleic acid, there are many kinds of chemical modification methods. The present invention adopts antisense nucleic acid modified by one or more combinations selected from ribose modification, base modification and phosphate backbone modification, and the pharmacology, pharmacokinetics, toxicity The preclinical research of science and other sciences is the most comprehensive research on various chemically modified antisense nucleic acids. The modified antisense nucleic acid can effectively prevent the antisense nucleic acid from being digested and degraded by a large number of exonucleases in the human body, thereby avoiding the antisense nucleic acid. The sense nucleic acid loses its proper biological activity. In addition, the thio-antisense nucleic acid can also stimulate the activity of RNase and degrade the RNA strand hybridized with it. Therefore, the antisense nucleic acid with these two modification methods is preferred to be used in experiments. It should be clear that any modification method that can increase the stability and bioavailability of the antisense nucleic acid can be used, such as cholesterol modification, PEG modification, etc. Preferably, the antisense nucleic acid modification of the present invention is selected from one or more of thio modification, 2'-methoxy modification and cholesterol modification. The most preferred modification method is as follows: all nucleotides are 2'-methoxy modified, and two nucleotides at the 5' end are modified with sulfur, and four nucleotides at the 3' end are modified with sulfur and at the 5' Or the 3' end is linked to cholesterol.

The antisense nucleic acid of the present invention has the effect of inhibiting the expression of miR-145. When the above antisense nucleic acid is transfected into the cell line U87/MG expressing miR-145, it can effectively inhibit the growth and malignant proliferation of U87/MG cells.

The present invention also provides a pharmaceutical composition, which contains a safe and therapeutically effective amount of the oligonucleotide of the present invention and a pharmaceutically acceptable carrier or excipient. Such delivery carriers include, but are not limited to, various carriers that can be used for nucleic acid delivery, such as liposomes, degradable polymer compounds, saline, buffer, glucose, water, glycerol, ethanol and combinations thereof. The pharmaceutical formulation should match the mode of administration.

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 a 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: the treatment of diseases related to the overexpression of miR-145 in the human body, including various solid tumors ( Especially glioma), various leukemias, etc.

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. Generally, the length of the antisense oligonucleotide is 13-35 bp, and for miRNA, it is preferably 18-23 bp.

Description of drawings

Figure 1 shows that miR-145 antisense oligonucleotides inhibit the expression of miR-145 in tumor cell U87/MG cells, and the three lines in Figure 1A-D are the results of repeated experiments. A is the expression of miR-145 after transfection of miR-145 antisense oligonucleotide (5'-AGGGAUUCCUGGGAAAACUGGAC-3'), B is the transfection of negative control antisense oligonucleotide (5'-CAGUACUUUUGUGUAGUACAA- 3') The expression of miR-145; C is the expression of the internal reference gene U6 after transfection of miR-145 antisense oligonucleotide; D is the expression of the internal reference gene U6 after transfection of the negative control antisense oligonucleotide E is the histogram of the inhibitory effect of miR-145 after transfection of antisense oligonucleotides, and the abscissa is the detected samples, where "inhibitor" means transfection of miR-145 antisense oligonucleotides The expression of miR-145 after acidification, "negative control" means the expression of miR-145 after transfection negative control.

Figure 2 shows that miR-145 antisense oligonucleotides inhibit the growth and proliferation of tumor cell U87/MG cells, A and B are U87/MG cells transfected with FAM-labeled negative controls; C is transfection negative The state of U87/MG cells after control; D is the state of U87/MG cells after transfection with miR-145 antisense oligonucleotide (5'-AGGGAUUCCUGGGAAAACUGGAC-3').

Detailed ways

The sequence of the antisense oligonucleotide of the present invention is complementary to 13-23 consecutive nucleotide sequences in 5'-GUCCAGUUUUCCCAGGAAUCCCU-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'-AGGGAUUCCUGGGAAAACUGGAC-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 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 very good inhibitory effect, the inhibition rate of miR-145 expression is more than 95%, and the inhibition rate of tumor cell growth is more than 65%.

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

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

Example 1, miR-145 antisense nucleic acid inhibits the expression of miR-145

For real-time quantitative fluorescence detection, the oligonucleotide sequence involved was synthesized by Shanghai Gemma Pharmaceutical Technology Co., Ltd. The specific experimental steps include:

Cell culture: U87/MG cells (purchased from the Cell Bank of the Type Culture Collection Committee of the Chinese Academy of Sciences), cultured in 10% FBS-DMEM medium (FBS was purchased from Hyclone, and DMEM was purchased from Gibco) at 37°C, 5% CO ₂ culture.

Cell transfection:

1) One day before transfection, inoculate cultured cells in a 24-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:

a. Use 50 μl serum-free Dilute miR-145 antisense oligonucleotide (5'-AGGGAUUCCUGGGAAAACUGGAC-3'), negative control (5'-CAGUACUUUUGUGUGUAGUACAA-3') and FAM-labeled negative control respectively in culture medium (Gibco) to a final concentration of 50 nM, Gently mix, set 3 replicate wells for each transfection;

培养基中，轻轻混匀后在室温下孵育5min；b. Gently mix Lipofecta mine ^TM 2000 (Invitrogen) before use, then take 2 μl and dilute to 50 μl

In the culture medium, mix gently and incubate at room temperature for 5 minutes;

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) Add the complex to each well containing cells and medium, and gently shake the culture plate back and forth to mix;

4) Incubate overnight at 37°C in a 5% CO ₂ incubator, replace the medium containing 10% fetal bovine serum and continue culturing for 24 hours.

Total RNA extraction:

1) Collect cells by centrifugation, add 500 μl Ezol (Invitrogen) to the centrifuge tube, invert the centrifuge tube up and down to mix, and place at room temperature for 10 min.

2) Add 200 μl of RNA-specific chloroform (Shanghai Sangong), and mix vigorously up and down until the liquid in the centrifuge tube is thoroughly mixed and becomes milky white.

3) Place at room temperature for 5 minutes, and centrifuge at 12000 rpm for 15 minutes.

4) Carefully transfer the supernatant to another clean 1.5ml centrifuge tube, avoiding aspiration of the middle protein phase and the lower organic phase.

5) Add 500 μl of pre-cooled RNA-specific isopropanol (Shanghai Sangong) to the supernatant, and let stand at room temperature for 5 minutes. Centrifuge at 10000rpm for 10min.

6) Discard the supernatant carefully, add 1ml of 75% ethanol (Shanghai Sangong) dedicated to RNA to wash the precipitate, and centrifuge at 10000rpm for 10min.

7) Discard the supernatant carefully, let the ethanol dry at room temperature, add 20 μl DEPC water to each tube to dissolve, and mix well.

RNA reverse transcription:

The RNA extracted above was reverse-transcribed with two RNA-specific reverse transcription primers, U6 and hsa-miR-145, respectively, to prepare cDNA templates. The buffers and enzymes used in reverse transcription were all produced by Promega; the primer sequences were synthesized by Shanghai Gemma Pharmaceutical Technology Co., Ltd., miR-145RT primer: 5'-GTCGGGTCCAGAGCAGGGTCCGAGGTACACGTTCGCTCTGGACCCGACAGGGATTCCT-3'.

(i). Reverse transcription system:

(ii). Reverse transcription reaction conditions:

The reaction conditions are: 26°C for 30 minutes; 42°C for 30 minutes; 85°C for 10 minutes.

Fluorescent quantitative PCR detection:

(1). Dilution of cDNA template:

Dilute the cDNA obtained after the above reverse transcription 3 times, add 40 μl RNase/DNase-free ddH ₂ O to the 20 μl system, and mix well.

(2). Fluorescence quantitative PCR system (primer sequence was synthesized by Shanghai Jima Pharmaceutical Technology Co., Ltd.; miR-145FP primer: 5'-TGCCGAGTCCAGTTTTTCCC-3'; miR-145RP primer: 5'-CAGAGCAGGGTCCGAGGTA-3'):

Reagent name Dosage/tube 2×PCR Master Mix 10μl F Primer (20μM) 0.2μl R Primer (20μM) 0.2μl template 2μl rTaq DNA polymerase (5U/ul) 0.2μl ddH ₂ O Add to 20μl

(3). Reaction conditions:

(i) 95°C for 3 minutes;

(ii) 95°C for 30s;

(iii) 62°C for 40s;

(iv) 72°C for 30s;

There are 40 cycles in total from steps ii to iv.

Fluorescent quantitative PCR detection results showed that antisense oligonucleotides had a good inhibitory effect. Accompanying drawing 1 shows that miR-145 antisense oligonucleotide inhibits the expression of miR-145 in tumor cell U87/MG cells, and the three lines in Fig. 1A-D are the results of repeated experiments. A is the expression of miR-145 after transfection of miR-145 antisense oligonucleotide (5'-AGGGAUUCCUGGGAAAACUGGAC-3'), B is the transfection of negative control antisense oligonucleotide (5'-CAGUACUUUUGUGUAGUACAA- 3') the expression of miR-145; C is the expression of the internal reference gene U6 after transfecting the miR-145 antisense oligonucleotide; D is the expression of the internal reference gene U6 after transfecting the negative control antisense oligonucleotide Expression status; E is the histogram of the inhibitory effect of miR-145 after transfection of antisense oligonucleotides, and the abscissa is the detected samples, where "inhibitor" means transfection of miR-145 antisense oligonucleotides The expression of miR-145 after transfection, "negative control" means the expression of miR-145 after transfection negative control. The results showed that antisense oligonucleotides had a significant inhibitory effect on the expression of miR-145.

Example 2. Detection of inhibitory activity of miR-145 antisense oligonucleotides on human glioma cell line U87/MG

The oligonucleotide sequences involved 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:

培养基(Gibco)分别稀释miR-145反义寡聚核苷酸(5‘-AGGGAUUCCUGGGAAAACUGGAC-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 25 μl serum-free

Dilute miR-145 antisense oligonucleotides (5'-AGGGAUUCCUGGGAAAACUGGAC-3'.), negative control (5'-CAGUACUUUUGUGUGUAGUACAA-3') and FAM-labeled negative control in 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;

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 0.25μl and dilute to 25μl

Culture medium, mix gently and incubate at room temperature for 5 minutes;

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 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 2, 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 transparent strong (Panel C); while most U87/MG cells died after transfection of miR-145 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 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 inhibitory rate of miR-145 antisense oligonucleotides to human glioblastoma cell lines was 66.41±4.56%. The results show that: the miR-145 antisense oligonucleotide provided by the invention has a good inhibitory effect, and the inhibitory rate on the growth of U87/MG exceeds 65%. The miR-145 antisense oligonucleotide of the present invention can effectively inhibit the expression of miR-145 in human brain glioma cells, inhibit its growth and proliferation, thereby effectively treating brain glioma and other tumors with high miR-145 expression.

Claims (10)

1. an antisense oligonucleotide is characterized in that, said antisense oligonucleotide comprise with 5 '-GUCCAGUUUUCCCAGGAAUCCCU-3 ' in continuous 13～23 nucleotide sequence complementary sequences.

2. antisense oligonucleotide as claimed in claim 1 is characterized in that said antisense oligonucleotide comprises sequence 5 '-AGGGAUUCCUGGGAAAACUGGAC-3 '.

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

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

5. antisense oligonucleotide as claimed in claim 4 is characterized in that, said 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, said modification is selected from one or more in thio-modification, 2 '-methoxyl group modification and the SUV modification.

7. antisense oligonucleotide as claimed in claim 6; It is characterized in that; All Nucleotide carry out 2 '-methoxyl group to be modified, and two Nucleotide of 5 ' end carry out thio-modification, and four Nucleotide of 3 ' end carry out thio-modification and connect SUV at 5 ' or 3 ' end.

8. be used to prepare the purposes of medicine of the relative disease of treatment miR-145 over-expresses like each described antisense oligonucleotide in the claim 1～7.

9. purposes as claimed in claim 8 is characterized in that, the relative disease of said miR-145 over-expresses is a cerebral glioma.

10. a pharmaceutical composition is characterized in that, contains each described antisense oligonucleotide and pharmaceutically acceptable carrier in the claim 1～7 of treating significant quantity.

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