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miRNA138 and application of miRNA138 in regulation of TERT gene expression

A technology of expression quantity and coding gene, applied to miRNA138 and its application in regulating TERT gene expression, can solve the problems of limited amplification ability, easy aging, and decreased regeneration ability.

Pending Publication Date: 2020-01-24
XINXIANG MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

EPCs have limited expansion ability under in vitro culture conditions, the regeneration ability decreases with the increase of differentiation times, and they are prone to aging.

Method used

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  • miRNA138 and application of miRNA138 in regulation of TERT gene expression
  • miRNA138 and application of miRNA138 in regulation of TERT gene expression
  • miRNA138 and application of miRNA138 in regulation of TERT gene expression

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1. Activity analysis of miRNA138 on the expression of luciferase reporter gene

[0066] Recombinant plasmid pEZX-MT01-3'UTR is the polycloning of TERT gene (sequence 3) inserted into pEZX-MT01 plasmid (purchased from Guangzhou Funeng Gene Co., Ltd., article number: CmiT000001-MT01) upstream of Renilla luciferase reporter gene hLuc The resulting plasmid between sites XhoI and EcoRI.

[0067] First transfect the Hela cells with the recombinant plasmid pEZX-MT01-3'UTR with the transfection reagent Lipofecta-mine 2000 to obtain the Hela cells transfected with pEZX-MT01-3'UTR, 6 hours later use the transfection reagent Lipofectamine RNAiMAX to transfect 50nM The miR138 solution (the solvent is water, the solute is miR138) was transfected into pEZX-MT01-3'UTR Hela cells for 48 hours to obtain pEZX-MT01-3'UTR+miR138-transfected Hela cells.

[0068] Microplate reader was used to detect the expression of luciferase reporter gene in Hela cells transfected with pEZX-MT01-...

Embodiment 2

[0070] Embodiment 2, the influence of miRNA138 on Hela cell TERT gene mRNA expression level

[0071] The effects of miRNA138 mimic and inhibitor on TERT gene mRNA levels in Hela cells were detected respectively, and no miRNA138 mimic and inhibitor were used as the control group.

[0072] Control group: Hela cells were cultured normally to 80% confluence.

[0073] miRNA group: after the hela cells were cultured to 80% confluence, they were transfected with 50 nM miRNA138 mimic solution (the solvent was water and the solute was miRNA138 mimic) with Lipofectamine RNAiMAX, and cultured for 48 hours.

[0074] miRNA inhibitor group: After cultured to 80% fusion, Lipofectamine RNAiMAX was used to transfect 50 nM miRNA138 inhibitor solution (solvent is water, solute is miRNA138 inhibitor), and cultured for 48 hours.

[0075] After 48 hours of transfection in the above-mentioned groups, the mRNA level of TERT gene in Hela cells was detected by PCR, and the primers used were as follows...

Embodiment 3

[0083] Example 3, the effect of miRNA138 on the angiogenesis ability of rat bone marrow-derived endothelial progenitor cells (EPCs)

[0084] 1. Isolation and identification of endothelial progenitor cells (EPC) derived from rat bone marrow

[0085] Femurs of adult SD rats (purchased from Guangdong Provincial Medical Experimental Animal Center, use license number: SYXK (Guangdong) 2010-0106) were taken under aseptic conditions, the bone marrow was washed out, and mononuclear cells were isolated. Suspension cells were cultured with EGM-2 medium, and unattached cells were discarded after 48 hours. Change the solution every 3 days. After 10-14 days when the cells are full, digest the cells and climb the slices for EPC immunofluorescence staining to identify the cultured EPCs ( Figure 4 ; A. Rat bone marrow mononuclear cells cultured in the Fn-coated culture dish in the endothelial cell culture system for 7 days to form adherent growth cells, which are egg-shaped or polygonal; B....

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Abstract

The invention discloses miRNA138 and an application of the miRNA138 in regulation of TERT gene expression. The invention provides miRNA138 and an application of an inhibitor of the miRNA138. The invention finds that the miR138 has a remarkable inhibition effect on the expression level of TERT mRNA of Hela cells, an oligonucleotide inhibitor of the miR-138 has a remarkable up-regulation effect on the mRNA expression level of the TERT gene of the Hela cell, the inhibitor of the miR-138 has the advantages that the blood vessel forming capability of the EPC is obviously improved, according to theinfluence of the miR-138 inhibitor on the differentiation of the EPC co-cultured NSC, the EPC transfected by the inhibitor of the miR-138 obviously inhibits the differentiation of the NSC to the glialcells.

Description

technical field [0001] 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 [0002] Vascular dementia (VD) refers to the clinical syndrome of cerebral intelligence and cognitive dysfunction caused by brain dysfunction caused by cerebrovascular disease, mainly manifested as learning, memory, thinking and other obstacles. It is the second leading cause of dementia after silent 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 rate of the elderly over 65 years old is 1%-4%, and the prevalence rate of the elderly aged 85 is as high as 14%- 16%. Alzheimer's disease, including VD, has become the fourth leading cause of death in the elderly after tumors, heart disease, and stroke. Therefore, finding anti-VD drugs and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113A61K31/7088A61K45/00A61K31/7105A61P35/00A61P25/28C12Q1/6883C12Q1/6886
CPCC12N15/1137A61K31/7088A61K45/00A61P35/00A61P25/28C12Q1/6883C12Q1/6886C12N2310/141C12Y207/07049C12Q2600/158C12Q2600/178C12Q2600/136
Inventor 王峰李娜娜王永玲张煜郭勇常连生张利彬魏茜茜董磊李昭一闫鹏云党沛余乐文范甜甜张素婷
Owner XINXIANG MEDICAL UNIV
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