Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Application of human microRNAs miR-185 to preparation of cell growth and/or senescence regulation agent

A technology of 1. mir-185 and regulator, applied in the field of biomedicine, can solve the problems of cell growth and senescence regulation without miRNAs, so as to promote the telomere disorder and senescence of tumor cells, inhibit the growth of tumor cells, and delay senescence. Effect

Inactive Publication Date: 2016-12-14
SUN YAT SEN UNIV
View PDF2 Cites 4 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are no studies and reports on miRNAs in the regulation of cell growth and aging, especially the regulation of POT1 expression

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Application of human microRNAs miR-185 to preparation of cell growth and/or senescence regulation agent
  • Application of human microRNAs miR-185 to preparation of cell growth and/or senescence regulation agent
  • Application of human microRNAs miR-185 to preparation of cell growth and/or senescence regulation agent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Construction of miR-185 overexpression cells and rescue (rescue) experiments

[0048] 1. Experimental method

[0049] Synthesize oligo primers containing the positive and negative sequences of shPOT1, anneal the two primers by gradient cooling, and ligate the annealed double-stranded RNA containing the positive and negative sequences of shPOT1 into the pLKO vector. HEK293T cells were transfected with plasmids expressing miRNA or shPOT1 to prepare viruses. Human fibrosarcoma cells HTC75 were infected with virus to construct a stable transfected cell line. Rescue experiment: The full-length GFP or POT1 was cloned into pBabe-CMV-DEST-SFB, transfected into HEK293T cells, and the virus was prepared. The miR-185 overexpression cell line was infected with virus to construct a rescue (rescue) cell line. Cell lines were tested for protein expression by immunoblotting.

[0050] 2. Experimental results

[0051] attached Figure 5 In B, the expression of POT1 in eac...

Embodiment 2

[0052] Example 2 Western blot experiment

[0053] 1. Experimental materials

[0054] Reagents: POT1 endogenous antibody used in this experiment (purchased from Novus Biologicals, product number NB500-176), diluted 1:1000 with 3% bovine serum albumin (BSA) before use; GAPDH antibody (purchased from Abmart company, product number 3B3), diluted with 3% BSA at 1:5000; secondary antibody (goat anti-mouse, purchased from LI-COR Company, product number 926-68050, goat anti-rabbit, purchased from LI-COR Company, product number 926- 32211), diluted 1:10000 with 3% BSA when used.

[0055] Human fibrosarcoma cell HTC75 was preserved by the Songyangzhou Research Group of the School of Life Sciences, Sun Yat-sen University.

[0056] 2. Experimental method

[0057] S1. Preparation of cell lysate: culture according to conventional human tumor cell culture methods, collect cells, wash once with phosphate buffered saline (PBS); suspend in PBS, add 5X SDS loading buffer to lyse and boil.

...

Embodiment 3

[0068] Example 3 Real-time fluorescent quantitative PCR experiment

[0069] 1. Experimental method

[0070] Total cellular RNA was extracted using TRIzol (Invitrogen, Cat. No. 15596-026), treated with DNase I (Invitrogen, Cat. No. 18068015) to remove residual DNA, and then reverse-transcribed into cDNA using TAKARA PrimeScript™ II1st Strand cDNA Synthesis Kit. Using SYBR Green (ABI, Cat. No. 4309155) as the dye, real-time fluorescent quantitative PCR experiments were performed. Set the PCR program: first step, 95°C for 10 minutes; second step: 95°C for 15 seconds, 60°C for 1 minute, 40 cycles. Melting curve program: Step 1, 95°C for 10 minutes; Step 2: 95°C for 10 seconds, 55°C for 10 seconds, each cycle increases 55°C by 0.5°C, and ends after rising to 95°C.

[0071] 2. Experimental results

[0072] The experimental results are attached image 3 A and Figure 5 As shown in A. attached image 3 A shows that in miR-185 overexpression cells, the mRNA level of POT1 is lowe...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention provides application of human microRNAs miR-185 to the preparation of a cell growth and / or senescence regulation agent. The miR-185 can directly act in a 3'-untranslated region (3'-UTR) of a messenger RNA (mRNA) of human telomere POT1, then mRNA and protein expression of POT1 can be inhibited, telomere damage and telomere length disorder can be caused, cell aging can be accelerated, and cell growth velocity can be inhibited. Therefore, miR-185 has significant regulation functions in the cell growth and aging process, effective cell growth and aging regulation agents can be developed for miR-185, for example, an anti-aging medicine can be developed and used in the cell aging delaying process, a tumor growth inhibitor can be developed for inhibiting growth of tumor cells, and the medicines can be used in anticancer treatment and all have great significance.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to the application of human microRNAs (microRNAs) miR-185 in the preparation of cell growth and aging regulators. Background technique [0002] Telomere is a highly ordered special structure composed of double-stranded TTAGGG repeat sequence and single-stranded 3'overhang composed of DNA sequence and its interacting proteins at the end of eukaryotic chromosome. It can protect chromosome ends, maintain genome stability and participate in biological processes such as cell aging regulation. Every time a normal human cell divides, the length of the telomere will gradually shorten due to the problem of terminal replication. When the telomere shortens to a certain limit, the cell will not be able to continue to divide and enter the process of aging and death. Telomere-binding protein is a special protein complex that specifically binds to telomeres. It has the functions ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): A61K45/00A61K31/7088A61P35/00A61P39/00
CPCA61K45/00A61K31/7088
Inventor 松阳洲黄燕黄军就李婷婷
Owner SUN YAT SEN UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com