MicroRNA328 for regulating expression of TERT gene and application of microRNA328

A technology of expression quantity and encoding gene, applied to microRNA328 regulating TERT gene expression and its application field, which can solve the problems of limited amplification ability, decreased regeneration ability, and easy aging.

Active Publication Date: 2019-12-31
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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  • MicroRNA328 for regulating expression of TERT gene and application of microRNA328
  • MicroRNA328 for regulating expression of TERT gene and application of microRNA328
  • MicroRNA328 for regulating expression of TERT gene and application of microRNA328

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Example 1. Activity analysis of miRNA328 on luciferase reporter gene expression

[0077] 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.

[0078] 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 miR328 solution (the solvent is water, the solute is miR328) was transfected into pEZX-MT01-3'UTR Hela cells for 48 hours to obtain pEZX-MT01-3'UTR+miR328-transfected Hela cells.

[0079] Microplate reader was used to detect the expression of luciferase reporter gene in Hela cells transfected with pEZX-MT01-3'UTR+m...

Embodiment 2

[0081] Embodiment 2, the influence of miRNA328 on Hela cell TERT gene mRNA level

[0082] The effects of miRNA328 mimic and inhibitor on TERT gene mRNA levels in Hela cells were detected respectively, and no miRNA328 mimic and inhibitor were added as the control group.

[0083] Control group: Hela cells were cultured normally to 80% confluence.

[0084] miRNA group: after the hela cells were cultured to 80% confluence, they were transfected with 50 nM miRNA328 mimic solution (the solvent was water and the solute was miRNA328 mimic) with Lipofectamine RNAiMAX, and cultured for 48 hours.

[0085] miRNA inhibitor group: After cultured to 80% fusion, Lipofectamine RNAiMAX was used to transfect 50 nM miRNA328 inhibitor solution (solvent is water, solute is miRNA328 inhibitor), and cultured for 48 hours.

[0086] 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:

[0087...

Embodiment 3

[0094] Example 3. Effect of miRNA328 on the angiogenesis ability of rat bone marrow-derived endothelial progenitor cells (EPCs)

[0095] 1. Isolation and identification of endothelial progenitor cells (EPC) derived from rat bone marrow

[0096] 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. Wrig...

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Abstract

The present invention discloses a microRNA328 for regulating expression of TERT gene and an application of the microRNA328, and provides uses of the microRNA328 or a substance inhibiting expression ofthe microRNA328. The miR328 is found to have a significant inhibitory effect on an expression level of TERT mRNA in Hela cells and an oligonucleotide inhibitor of the miR-328 has a significant up-regulation effect on the expression level of the TERT gene mRNA in the Hela cells. The inhibitor of the miR-328 significantly increases angiogenic capacity of EPC. The EPC transfected by the inhibitor ofthe miR-328 significantly inhibits differentiation of NSCs into glial cells. Studies show that using miRNAs as targets or tools to enhance telomerase activity provide theoretical basis for treatmentof neurological diseases of vascular dementia (VD), etc. and have potential clinical application value.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to microRNA328 regulating TERT gene expression and application thereof. 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 an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K31/7105A61P9/00A61P25/00A61P25/28A61P35/00
CPCA61K31/7105A61P9/00A61P25/00A61P35/00A61P25/28
Inventor 王峰王永玲李娜娜张煜刘淑媛张利彬常连生魏茜茜李昭一董磊闫鹏云董子源安波涛尹朝华
Owner XINXIANG MEDICAL UNIV
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