MicroRNA328 regulating tert gene expression and its applications

A technology of encoding genes and inhibitors, applied to microRNA328 that regulates the expression of TERT gene and its application fields, can solve the problems of easy aging, limited amplification ability, and decreased regeneration ability

Active Publication Date: 2022-07-05
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 regulating tert gene expression and its applications
  • MicroRNA328 regulating tert gene expression and its applications
  • MicroRNA328 regulating tert gene expression and its applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0077] 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.

[0078] First, the recombinant plasmid pEZX-MT01-3'UTR was used to transfect Hela cells with the transfection reagent Lipofecta-mine 2000 to obtain Hela cells transfected with pEZX-MT01-3'UTR. The miR328 solution (solvent is water, solute is miR328) was transfected into Hela cells of pEZX-MT01-3'UTR for 48h to obtain Hela cells transfected with pEZX-MT01-3'UTR+miR328.

[0079] The expression of luciferase reporter gene in Hela cells transfected with pEZX-MT01-3'UTR+miR328 was detected by microplate reader, Renilla luciferase gene was used as reporter gene, and firefly luciferase gen...

Embodiment 2

[0081] Example 2. The effect of miRNA328 on the mRNA level of TERT gene in Hela cells

[0082] The mimic and inhibitor of miRNA328 were used to detect the effect on the mRNA level of TERT gene in Hela cells, and the mimic and inhibitor without any miRNA328 were used as the control group.

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

[0084] miRNA group: hela cells were cultured to 80% confluency and then transfected with 50nM mimic solution of miRNA328 with Lipofectamine RNAiMAX (solvent was water, solute was mimic of miRNA328), and cultured for 48h.

[0085] miRNA inhibitor group: cultured to 80% confluence, transfected with Lipofectamine RNAiMAX into 50nM inhibitor solution of miRNA328 (solvent is water, solute is inhibitor of miRNA328), and cultured for 48h.

[0086] 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:

[0087] TERT upstream primer: 5'AGCATTTCAC...

Embodiment 3

[0094] Example 3. The effect of miRNA328 on the angiogenesis of rat bone marrow-derived endothelial progenitor cells (EPC)

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

[0096] Under sterile conditions, the femurs of adult SD rats (purchased from the Guangdong Provincial Medical Laboratory Animal Center, license number: SYXK (Guangdong) 2010-0106) were taken, the bone marrow was flushed out, and the 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 EPC ( Figure 4 ; A. Rat bone marrow mononuclear cells were cultured in Fn-coated culture dish for 7 days and formed adherent growth cells, which were oval or polygonal; B. Wright's Giemsa staining for adherent cells (×200)), wh...

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Abstract

The invention discloses microRNA328 regulating TERT gene expression and its application. The present invention provides the use of microRNA328 or a substance that inhibits its expression. The present invention finds that miR328 has a significant inhibitory effect on the expression level of TERT mRNA in Hela cells, and an oligonucleotide inhibitor of miR-328 has a significant up-regulation effect on the expression level of TERT gene mRNA in Hela cells. The inhibitor of miR-328 significantly increased the angiogenic capacity of EPCs. EPCs transfected with the inhibitor of miR-328 significantly inhibited the differentiation of NSCs into glial cells. Studies have shown that using miRNA as a target or tool to enhance the activity of telomerase provides a theoretical basis for the treatment of neurological diseases such as vascular dementia (VD), and has potential clinical application value.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to microRNA328 regulating TERT gene expression and its application. Background technique [0002] 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 obstacles. 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 wo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113A61K31/7105A61P9/00A61P25/00A61P25/28A61P35/00
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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