CN110724688A - miRNA138 and its application in regulating TERT gene expression - Google Patents

Abstract

The invention discloses miRNA138 and application thereof in regulating TERT gene expression. The invention provides miRNA138 and uses of inhibitors thereof. The invention discovers that miR138 has a remarkable inhibiting effect on the expression level of TERT mRNA of Hela cells, an oligonucleotide inhibitor of miR-138 has a remarkable up-regulating effect on the expression level of TERT gene mRNA of Hela cells, the inhibitor of miR-138 has the capability of remarkably increasing the angiogenisis of EPC, the miR-138inhibitor has the influence on the differentiation of NSC in EPC co-culture, and the EPC transfected by the inhibitor of miR-138 obviously inhibits the differentiation of NSC to glial cells.

Description

miRNA138 and its application in regulating TERT gene expression

technical field

The invention belongs to the field of biotechnology, and in particular relates to a miRNA138 and its application in regulating TERT gene expression.

Background technique

Vascular dementia (VD) is a clinical syndrome caused by brain dysfunction caused by cerebrovascular disease, resulting in brain intelligence and cognitive dysfunction, mainly manifested as learning, memory, thinking and other impairments. It is the second leading cause of dementia after Alzheimer's disease (AD). With the acceleration of the aging process of the world population, the number of patients with VD has increased significantly. According to epidemiological statistics, the prevalence of elderly people over 65 years old is 1%-4%, and the prevalence of elderly people aged 85 years is as high as 14%- 16%. Alzheimer's disease including VD has become the fourth leading cause of death among the elderly after tumor, heart disease and stroke. Therefore, it is an urgent task for medical workers to find anti-VD drugs and treatment methods.

The discovery of neural stem cells (NSC) has brought new hope for the repair of nerve damage. Some studies have also shown that the co-culture of EPC and NSC can provide a good microenvironment for neurogenesis, improve the proliferation of NSC and promote It differentiates into neurons. The EPC activity and quantity are related to EPC senescence. Cellular senescence is due to cell cycle arrest, and telomere shortening during cell replication is the most common cause of senescence. Telomeres are composed of DNA and proteins, located at the ends of eukaryotic chromosomes, and their DNA is made up of specific repeating sequences. DNA repeats are expressed differently across species. With the replication of chromosomes, the length of telomeres will shorten, but after shortening to a certain length, it will not shorten further, and cells can no longer divide, showing changes in senescence and apoptosis. Telomerase is a ribonucleoprotein that consists of 3 parts: telomerase RNA (telomerase RNA, hTR or TRC) template, telomerase-related protein (TPI) and telomerase reverse transcriptase (TERT) , has the activity of regulating telomere length, function and reverse transcription. TERT, one of the protein components of telomerase, is the catalytic subunit of telomerase and plays an important role in the activation of telomerase and the specific amplification of telomere DNA. TERT can inhibit apoptosis by affecting telomerase activity and further extending telomere length. When TERT is highly expressed, it can activate telomerase activity, inhibit telomere shortening or slow down the speed of telomere shortening or even promote telomere lengthening, and achieve the effect of anti-aging and apoptosis. EPCs have the characteristics of limited expansion ability under in vitro culture conditions, decreased regeneration ability with the increase of differentiation times, and easy aging. Therefore, regulating EPC telomerase activity to prevent EPC senescence and enhance its in vitro and in vivo proliferation and angiogenesis functions are issues worth pondering and exploring.

miRNAs are a class of highly conserved single-stranded non-coding small RNAs, consisting of about 20-22 single nucleotides, widely present in eukaryotes and involved in post-transcriptional regulation.

SUMMARY OF THE INVENTION

In order to treat diseases caused by increased or decreased expression of TERT protein or gene, the present invention provides the following technical solutions:

An object of the present invention is the use of a substance that inhibits the expression of microRNA138 or its encoded gene.

Application of the substance that inhibits the expression of microRNA138 or its encoded gene provided by the present invention in at least one of the following A1)-A5) or in the preparation of a product with at least one of the following functions in A1)-A5):

A1) Improve the activity of TERT protein or the expression of gene encoding TERT protein;

A2) increase the TERT protein activity or the expression of the TERT protein-encoding gene in cells;

A3) Treatment of diseases caused by decreased TERT protein activity or decreased expression of TERT protein-encoding genes;

A4) Improve the angiogenesis ability of endothelial progenitor cells;

A5) Inhibit the differentiation of neural stem cells to glial cells;

The microRNA138 is as follows B1) or B2):

B1) nucleic acid shown in sequence 1;

B2) A nucleic acid molecule with the same function as Sequence 1, which has undergone one or several nucleotide substitutions and/or deletions and/or additions.

The above-mentioned substitutions and/or deletions and/or additions of one or several nucleotides are substitutions and/or deletions and/or additions of no more than 10 nucleotides.

In the above application, the substance is a microRNA138 inhibitor or a microRNA138 inhibitory oligonucleotide. In the embodiment of the present invention, the microRNA138 inhibitory oligonucleotide is the nucleotide sequence shown in sequence 2 (an inhibitor of miRNA138), which was purchased from Guangzhou RiboBio Technology Co., Ltd. Ruibo Company, it is well known that this sequence is an inhibitor of miRNA138.

In the above application, in A2, the cells are tumor cells or neural stem cells;

The tumor cells are specifically cervical cancer cells, which are hela cells in the embodiment of the present invention;

The second object of the present invention is to provide a substance that inhibits the expression of microRNA138 or its encoding gene.

A substance that inhibits the expression of microRNA138 or its encoded gene provided by the present invention is a microRNA138 inhibitor or a microRNA138 inhibitory oligonucleotide

In the embodiment of the present invention, the above-mentioned microRNA138 inhibitory oligonucleotide is the following C1) or C2):

C1) is the nucleic acid shown in sequence 2;

C2) is a nucleic acid molecule in which sequence 2 is substituted and/or deleted and/or added by one or several nucleotides and has the same function as sequence 1;

C3) Recombinant vectors, recombinant microorganisms or transgenic cells containing the genes encoding the nucleic acids shown in C1) or C2).

The third object of the present invention is to provide a method for increasing the activity of TERT protein or the expression of TERT protein-encoding gene in cells.

The method provided by the present invention includes the following steps: inhibiting the expression of microRNA138 or the gene encoding it in the cell, so as to improve the activity of the TERT protein or the expression of the gene encoding the TERT protein in the cell.

The fourth object of the present invention is to provide a product comprising the above-mentioned substances that inhibit the expression of microRNA138 or its encoded gene;

The product has at least one function in the following A1)-A5):

A1) Improve the activity of TERT protein or the expression of gene encoding TERT protein;

A2) increase the TERT protein activity or the expression of the TERT protein-encoding gene in cells;

A3) Treatment of diseases caused by decreased TERT protein activity or decreased expression of TERT protein-encoding genes;

A4) Improve the angiogenesis ability of endothelial progenitor cells;

A5) Inhibit the differentiation of neural stem cells into glial cells.

The above inhibition of neural stem cells from differentiation into glial cells inhibits neural stem cells from differentiated into glial cells when co-cultured with endothelial progenitor cells and neural stem cells.

The fifth object of the present invention is to provide the use of microRNA138 or its encoding gene or a recombinant vector, recombinant microorganism or transgenic cell containing the encoding gene.

The present invention provides microRNA138 or its encoding gene or a recombinant vector containing its encoding gene, a recombinant microorganism or a transgenic cell in at least one of the following D1)-D5) or prepared to have at least one of the following functions in D1)-D5). The application of the product:

D1) reducing TERT protein activity or expression of TERT protein-encoding genes;

D2) reducing TERT protein activity or expression of TERT protein-encoding genes in cells;

D3) Treating diseases caused by increased TERT protein activity or increased expression of TERT protein-encoding genes;

D4) reduce the angiogenesis ability of endothelial progenitor cells;

D5) promote the differentiation of neural stem cells to glial cells;

The microRNA138 is as follows B1) or B2):

B1) nucleic acid shown in sequence 1;

B2) A nucleic acid molecule with the same function as Sequence 1, which has undergone one or several nucleotide substitutions and/or deletions and/or additions.

When the neural stem cells are promoted to differentiate into glial cells into endothelial progenitor cells and the neural stem cells are co-cultured, the neural stem cells are promoted to differentiate into glial cells.

The sixth object of the present invention is to provide a method for reducing TERT protein activity or expression of TERT protein-encoding genes in cells.

The method for the purpose of the present invention includes the following steps: increasing the expression of microRNA138 or its encoding gene in the cell, and reducing the TERT protein activity or the expression of the TERT protein encoding gene in the cell.

The application of miRNA138 as a target or tool in the preparation or screening of drugs for diseases caused by decreased or increased expression of TERT protein or its gene is also within the scope of protection of the present invention.

The present invention seeks out miRNAs that are highly expressed in the EPC of endothelial progenitor cells and specifically bind to the action site in the TERT gene mRNA sequence, and then use an inhibitor to combine with miRNAs to prevent it from binding to the action site of TERT, thereby up-regulating TERT and co-transplanted with NSCs into the rat model of vascular dementia, inducing the differentiation of NSCs into neurons, providing a theoretical basis for the treatment of neurological diseases such as vascular dementia, and having potential clinical application value.

The inventors found that miR138 significantly inhibited the expression level of TERT mRNA in Hela cells. Oligonucleotide inhibitors of miR-138 significantly up-regulated the expression level of TERT gene mRNA in Hela cells. In order to verify the effect of miR-138 on the angiogenesis ability of EPC, the experimental results showed that the inhibitory effect of miR-138 was observed in this experiment, and it was found that it significantly increased the angiogenesis ability of EPC. Compared with the control group, the tubular structure increased to (8.67 ±1.53). The experiment further observed the effect of miR-138 inhibitor on the differentiation of NSCs co-cultured with EPCs. EPCs transfected with miR-138 inhibitor significantly inhibited the differentiation of NSCs into glial cells.

Description of drawings

Figure 1 shows that Hela cells were transiently transfected with pEZX-MT01-TERT-3'UTR plasmid to analyze microRNA inhibitory activity.

Figure 2 shows the effect of microRNA on TERT mRNA level.

Figure 3 shows the effect of microRNA oligonucleotide inhibitors on TERT mRNA levels.

Figure 4 shows the morphological observation of rat bone marrow-derived endothelial cells; A. The adherent growth cells formed by rat bone marrow mononuclear cells cultured in the Fn-coated culture dish for 7 days in the endothelial cell culture system are oval or polygonal ; B. Wright's Giemsa staining for adherent cells (×200).

Figure 5 shows the immunochemical staining of rat bone marrow mononuclear cells after being cultured in endothelial cell culture system. A, B, and C are the expressions of vWF, CD31, and CD144, respectively, and D is the negative control staining of CD31 in fibroblasts (×200)

Figure 6 is the detection of EPC characteristics; A. The uptake of DiI-Ac-LDL by paving stone-like cells after culturing bone marrow mononuclear cells (×100); B. Detection of the ability to bind phytohemagglutinin UEA-1 (×200).

Figure 7 shows the effects of mimic and inhibitor of microRNA on the angiogenesis ability of EPCs (×100). A and B are microRNA mimic (Control, miR-138) transfection EPC angiogenesis observation. C and D are microRNA inhibitors (Control, miR-138) transfected into EPC to observe the angiogenesis ability. E. Statistics of blood vessel data of each composition. **P<0.01, compared with the corresponding mimic group. #P<0.5, compared with control inhibitor.

Figure 8 shows the effect of immunofluorescence detection of microRNA on the differentiation of EPC co-cultured NSCs (×100); A. Control group. B. EPC+NSC group. C. EPC/miRNA+NSC group. D.EPC/inhibitor+NSC group. Blue is DAPI nuclear staining of NSC, red is GFAP (glial fibrillary acidic protein) staining. E. Statistics of blood vessel data of each composition. **P<0.01, compared to EPC/miRNA+NSC. #P<0.5, compared with control and EPC+NSC.

Figure 9 shows the effect of miRNA138 on the expression of TERT protein in EPC co-cultured NSCs (*: P<0.05, compared with control).

Detailed ways

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

The mimic of miRNA138 in the following examples is the nucleotide sequence of miRNA138 shown in sequence 1;

The inhibitor of miRNA138 in the following examples is the nucleotide sequence shown in sequence 2, which was purchased from Guangzhou Ribo Biotechnology Co., Ltd., and it is known that this sequence is an inhibitor of miRNA138).

Example 1. Activity analysis of miRNA138 on luciferase reporter gene expression

The recombinant plasmid pEZX-MT01-3'UTR is a polyclonal upstream of the Renilla luciferase reporter gene hLuc by inserting the TERT gene (sequence 3) into the pEZX-MT01 plasmid (purchased from Guangzhou Funeng Gene Co., Ltd., item number: CmiT000001-MT01). Plasmid obtained between sites XhoI and EcoRI.

First, the recombinant plasmid pEZX-MT01-3'UTR was transfected into Hela cells with the transfection reagent Lipofecta-mine 2000 to obtain Hela cells transfected with pEZX-MT01-3'UTR. miR138 solution (solvent is water, solute is miR138) was transfected into pEZX-MT01-3'UTR Hela cells for 48h to obtain pEZX-MT01-3'UTR+miR138-transfected Hela cells.

The expression of luciferase reporter gene in Hela cells transfected with pEZX-MT01-3'UTR+miR138 was detected by microplate reader, Renilla luciferase gene was used as reporter gene, and firefly luciferase gene was used as normalization internal reference to analyze miR138 effect on the gene of interest.

The results are shown in Figure 1. Compared with other miRNAs, it was found that 50nM of miR138 showed a significant inhibitory effect on the luciferase gene expression activity in the transient transfection of pEZX-MT01-3'UTR reporter gene plasmid (p<0.01). It indicated that miR138 could inhibit the expression of TERT gene mRNA in Hela cells.

Example 2. The effect of miRNA138 on the expression level of TERT gene mRNA in Hela cells

The mimic and inhibitor of miRNA138 were used to detect the effect on the mRNA level of TERT gene in Hela cells, and the mimic and inhibitor of miRNA138 without any miRNA138 were used as the control group.

Control group: hela cells were cultured to 80% confluence.

miRNA group: hela cells were cultured to 80% confluency and then transfected with 50nM mimic solution of miRNA138 with Lipofectamine RNAiMAX (solvent was water, solute was mimic of miRNA138), and cultured for 48h.

miRNA inhibitor group: cultured to 80% confluence, transfected with Lipofectamine RNAiMAX into 50nM inhibitor solution of miRNA138 (solvent is water, solute is inhibitor of miRNA138), and cultured for 48h.

PCR detection of the mRNA levels of TERT gene in Hela cells 48 hours after transfection in the above groups, the primers used are as follows:

TERT upstream primer: 5'AGCATTTCACCCAGCGTCTA3'

Downstream primer: 5'CTTCAACCGCAAGACCGACA3'

PCR amplification conditions were: 94°C for 5 min after pre-denaturation, 40 cycles (94°C for 30s, 57°C for 30s, 72°C for 60s), followed by 72°C for 10 min and 4°C for 1 h.

The TERT gene expression in the control group was taken as 1, and the TERT gene expression in the other groups was the result relative to the control group.

The detection results of TERT gene mRNA levels in Hela cells under the action of miR138 are shown in Figure 2 (the change in TERT gene mRNA expression on the ordinate is the change in TERT gene expression relative to the control group). It can be seen that compared with the control group , the mRNA level of TERT gene in Hela cells decreased by 0.49 under the mimic effect of miR-138.

The results of the detection of TERT gene mRNA level in Hela cells under the action of the inhibitor of miR138 are shown in Figure 3; it can be seen that compared with the control group, the inhibitor of miR138 has a significant up-regulation effect on the mRNA expression level of the TERT gene in Hela cells. In contrast, the mRNA level of TERT gene in Hela cells could be increased to 1.12 under the inhibitor effect of miR138.

The above results show that miR-138 can inhibit the expression level of TERT gene mRNA in Hela cells, and miR-138 oligonucleotide inhibitor (miR138 inhibitor) can significantly up-regulate the effect of TERT gene mRNA expression in Hela cells.

Example 3. The effect of miRNA138 on the angiogenesis of rat bone marrow-derived endothelial progenitor cells (EPC)

1. Isolation and identification of rat bone marrow-derived endothelial progenitor cells (EPC)

The femurs of adult SD rats (purchased from Guangdong Provincial Medical Laboratory Animal Center, license number: SYXK (Guangdong) 2010-0106) were taken under sterile conditions, and the bone marrow was flushed out, and mononuclear cells were isolated. The cells were cultured in suspension in EGM-2 medium, and the non-adherent cells were discarded for 48 h. The medium was changed every 3 days, and after 10-14 days of cell growth, the cells were digested and stained for EPC immunofluorescence to identify the cultured EPCs (Fig. 4; A. Rat bone marrow mononuclear cells were placed in the Fn-coated culture dish in The adherent growing cells formed after 7 days of culture in the endothelial cell culture system were oval or polygonal; B. Wright's Giemsa staining for adherent cells (×200)); EPCs were detected for CD34, KDR, CD31, VE- The expression of cadherin surface antigen (Figure 5); detection of FITC-UEA-1 and acetylated low-density lipoprotein (DiI-Ac-LDL) phagocytosis, migration and angiogenesis (Figure 6); , EPCs with high proliferative potential, and provide a rich source of cells for further research.

2. The effect of miRNA138 on the angiogenesis ability of rat bone marrow-derived endothelial progenitor cells (EPC)

The EPC cells obtained in the above 1 were plated on a 12-well plate. After the cells had grown to 80%, the mimic of miRNA138 and the miRNA138 inhibitor were transferred into the cells respectively, and the medium was changed for 6 hours after transfection.

The above transfections are divided into the following groups:

Mimic (Control) of microRNA transfected EPC: 50nM Mimic solution of Control (solvent is water, solute is Mimic of Control) (mimic of Control, Guangzhou Ribo Biotechnology Co., Ltd., catalog number: miR1N0000001-1-5, Sequence 5'-UUUGUACUACACAAAAGUACAUG-3') was transfected into EPC to obtain EPC transfected with mimic (Control);

Mimic transfection of microRNA138 into EPC: Transfect 50nM mimic solution of microRNA138 (solvent is water, solute is mimic of microRNA138) to transfect EPC to obtain EPC transfected with microRNA138 mimic;

Inhibitor (Control) of microRNA is transfected into EPC: Transfect EPC with 50nM inhibitor solution of Control (solvent is water, solute is inhibitor of Control) (the sequence of inhibitor of Control: 5'-CGGCCTGATTCACAACACCAGCT-3') to obtain transfection inhibitor (Control) EPC;

Inhibitor transfection of microRNA138 into EPC: Transfect EPC with 50nM inhibitor solution of microRNA138 (solvent is water, solute is inhibitor of microRNA138) to obtain EPC transfected with microRNA138 inhibitor.

On the second day (24 hours after transfection), the EPCs transfected with various substances in the above groups were digested, firstly stained with calcein 5uM for 15min, and then the digested EPCs transfected with various substances (10,000/well) were digested. Seed in a 96-well plate coated with Matrigel (50ul), a total of 3 replicate wells; the formation of tubular structures was observed after incubation for 20 hours.

The results are shown in Figure 7, A and B are the angiogenesis ability of EPCs transfected with microRNA-transfected mimic (Control, miR-138); C, D are the angiogenesis ability of EPCs transfected with microRNA-transfected inhibitor (Control, miR-138). Observed. E. Statistics of blood vessel data of each composition. **P<0.01, compared with the control mimic group, the mimic of miR-138 significantly reduced the angiogenesis ability of EPC; #P<0.5, compared with the control inhibitor, the inhibitor of miR-138 significantly increased the angiogenesis of EPC Ability, tubular structure increased to 9.33 ± 2.08 compared to the control group.

Example 4. The effect of miRNA138 on EPC co-culture NSC differentiation

1. The effect of miRNA138 on the differentiation of EPC co-cultured NSCs

The EPCs transfected with various substances in 2 of Example 3 were incubated for 20 hours and then mixed with NSC cells to form a single cell suspension. The NSC cells were seeded in the lower layer of Transwell at a density of 1×10 ⁶ cells/cm ² and incubated for 20 hrs. The EPCs transfected with various substances were inoculated in the upper layer of Transwell at the same density) after co-cultivation for 7 days, and immunofluorescence detection was carried out, as follows:

The above co-cultures were divided into the following groups:

A. Control group: After 7 days of NSC cell culture, immunofluorescence detection was performed.

B. EPC+NSC group: After 7 days of co-culture of EPC and NSC cells (the number ratio of the two cells is 1:1), immunofluorescence detection was performed.

C. EPC/miRNA138+NSC group: microRNA138-transfected mimic EPC and NSC cells (the number ratio of the two cells was 1:1) were co-cultured for 7 days, and immunofluorescence detection was performed.

D. EPC/inhibitor+NSC group: EPC and NSC cells transfected with inhibitor of microRNA138 (the number ratio of the two cells was 1:1) were co-cultured for 7 days, and then immunofluorescence detection was performed.

The method of immunofluorescence detection is as follows: after fixing the above NSC cells with 4% paraformaldehyde, add GFAP primary antibody (purchased from Cell Signaling Technology, product number: 3670S) and incubate, overnight at 4°C, wash three times with PBS, and then add two Antibodies (purchased from Abcam, product number: ab150079)), incubated in a dark box at room temperature for 1 h, washed 3 times with PBS, mounted, observed under a fluorescence microscope and photographed.

The results are shown in Figure 8, A. control group; B. EPC+NSC group, C. EPC/miR-138+NSC group, D. EPC/inhibitor+NSC group. Blue is DAPI nuclear staining of NSC, red is GFAP (glial fibrillary acidic protein) staining; E. Statistics of blood vessels in each composition, **P<0.01, compared with EPC/miRNA+NSC, #P<0.5, compared with control and EPC+NSC comparison; it can be seen that: EPC/inhibitor+NSC group has significant difference compared with other groups, suggesting that EPC transfected with inhibitor of miR-138 significantly inhibits the differentiation of NSC into glial cells.

It was shown that the inhibitor of miR-138 inhibited the differentiation of NSCs into glial cells when EPC and NSC cells were co-cultured, and the mimic of miR-138 promoted the differentiation of NSCs into glial cells when EPC and NSC cells were co-cultured.

2. The effect of miRNA138 on the expression of TERT protein in EPC co-cultured NSCs

The EPCs transfected with various substances in Example 3 were incubated for 20 hours and co-cultured with NSCs (the ratio of the numbers of the two cells was 1:1) for 7 days, and then an enzyme-linked immunization (ELISA) experiment was performed, as follows:

The above co-cultures were divided into the following groups:

Mimic group of microRNA138: EPC and NSC cells transfected with mimic of microRNA138 were co-cultured for 7 days, and then the enzyme-linked immunosorbent assay (ELISA) experiment was performed.

The mimic group of control: EPC and NSC cells transfected with mimic of mcontrol were co-cultured for 7 days, and then the enzyme-linked immunosorbent assay (ELISA) was performed.

Inhibitor group of microRNA138: EPC and NSC cells transfected with inhibitor of microRNA138 were co-cultured for 7 days, and then the enzyme-linked immunosorbent assay (ELISA) experiment was performed.

Control inhibitor group: EPC and NSC cells transfected with control inhibitor were co-cultured for 7 days, and then an enzyme-linked immunosorbent assay (ELISA) was performed.

The method of enzyme-linked immunosorbent assay (ELISA) is as follows: extract the protein of NSC cells in the lower layer of Transwell with protein lysate containing 1% PMSF, and measure the protein content of each group according to the BCA protein assay instructions. The above-mentioned protein samples of each group were diluted 4 times, and the content of TERT in each group was determined according to the product instructions of the ELISA kit. Add 100 μl of the sample to be tested to each well of the sample to be tested, and set 3 duplicate wells in parallel. The optical density (OD) value of each well was measured with a microplate reader at a wavelength of 450 nm. The linear regression equation of the standard curve is obtained according to the concentration and OD value of the standard substance, and the sample concentration is calculated, and then multiplied by the dilution factor, which is the TERT concentration in the sample, and the TERT protein content in the sample is calculated.

The results are shown in Figure 9. Compared with the control group, the mimic EPCs transfected with miR-138 significantly inhibited the expression level of TERT protein in NSCs (P<0.001); while the EPCs transfected with miR-138 inhibitor were compared with the control group. The expression level of TERT protein in NSC cells was significantly up-regulated (P<0.01).

SEQUENCE LISTING

<110> Xinxiang Medical College

<120> miRNA138 and its application in regulating TERT gene expression

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<170> PatentIn version 3.5

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cagatgccgg cccacggcct attcccctgg tgcggcctgc tgctggatac ccggaccctg 2820

gaggtgcaga gcgactactc cagctatgcc cggacctcca tcagagccag tctcaccttc 2880

aaccgcggct tcaaggctgg gaggaacatg cgtcgcaaac tctttggggt cttgcggctg 2940

aagtgtcaca gcctgtttct ggatttgcag gtgaacagcc tccagacggt gtgcaccaac 3000

atctacaaga tcctcctgct gcaggcgtac aggtttcacg catgtgtgct gcagctccca 3060

tttcatcagc aagtttggaa gaaccccaca tttttcctgc gcgtcatctc tgacacggcc 3120

tccctctgct actccatcct gaaagccaag aacgcaggga tgtcgctggg ggccaagggc 3180

gccgccggcc ctctgccctc cgaggccgtg cagtggctgt gccaccaagc attcctgctc 3240

aagctgactc gacaccgtgt cacctacgtg ccactcctgg ggtcactcag gacagcccag 3300

acgcagctga gtcggaagct cccggggacg acgctgactg ccctggaggc cgcagccaac 3360

ccggcactgc cctcagactt caagaccatc ctggactga 3399

Claims (9)

1. The application of the substance for inhibiting the expression of the microRNA138 or the coding gene thereof in at least one of the following A1) -A5) or in the preparation of a product with at least one function of the following A1) -A5):

A1) increasing the TERT protein activity or expression of a TERT protein coding gene;

A2) increasing the activity of a TERT protein or the expression of a TERT protein coding gene in a cell;

A3) treating a disease caused by a decrease in the activity of a TERT protein or a decrease in the expression of a TERT protein-encoding gene;

A4) increasing the angiogenic capacity of endothelial progenitor cells;

A5) inhibiting differentiation of neural stem cells into glial cells;

the microRNA138 is B1) or B2) as follows:

B1) a nucleic acid as shown in sequence 1;

B2) and (b) the nucleic acid molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 1 and has the same function as the sequence 1.

2. Use according to claim 1, characterized in that:

the substance is a microRNA138 inhibitor or microRNA138 inhibitory oligonucleotide.

3. Use according to claim 1 or 2, characterized in that:

a2, wherein the cell is tumor cell or nerve stem cell;

the tumor cell is specifically a cervical cancer cell.

4. A substance for inhibiting the expression of microRNA138 or a coding gene thereof is a microRNA138 inhibitor or a microRNA138 inhibitory oligonucleotide.

5. A method for increasing TERT protein activity or expression of a TERT protein-encoding gene in a cell, comprising the steps of: and the expression of microRNA138 or the coding gene thereof in the cell is inhibited, so that the activity of the TERT protein or the expression of the TERT protein coding gene in the cell is improved.

6. A product comprising the substance of claim 4;

the product has at least one of the following functions A1) -A5):

A1) increasing the TERT protein activity or expression of a TERT protein coding gene;

A2) increasing the activity of a TERT protein or the expression of a TERT protein coding gene in a cell;

A3) treating a disease caused by a decrease in the activity of a TERT protein or a decrease in the expression of a TERT protein-encoding gene;

A4) increasing the angiogenic capacity of endothelial progenitor cells;

A5) inhibiting differentiation of neural stem cells into glial cells.

The application of microRNA138 or a coding gene thereof or a recombinant vector, a recombinant microorganism or a transgenic cell containing the coding gene thereof in at least one of D1) -D5) or in a product with at least one function of D1) -D5) as follows:

D1) reducing the TERT protein activity or expression of a TERT protein encoding gene;

D2) reducing TERT protein activity or expression of a TERT protein encoding gene in a cell;

D3) treating a disease caused by an increase in the activity of a TERT protein or an increase in the expression level of a TERT protein-encoding gene;

D4) reducing the angiogenic capacity of endothelial progenitor cells;

D5) promoting the differentiation of the neural stem cells to the glial cells;

the microRNA138 is B1) or B2) as follows:

B1) a nucleic acid as shown in sequence 1;

B2) and (b) the nucleic acid molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 1 and has the same function as the sequence 1.

8. A method of reducing TERT protein activity or expression of a TERT protein-encoding gene in a cell, comprising the steps of: improving the expression of microRNA138 or the coding gene thereof in the cell, and realizing the reduction of the activity of the TERT protein or the expression of the TERT protein coding gene in the cell.

The application of miRNA138 as a target or tool in preparing or screening medicines for diseases caused by reduction or improvement of TERT protein or gene expression thereof.

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