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Rat long-chain non-coding lncRNA-lncMSTRG10078 and application of rat long-chain non-coding lncRNA-lncMSTRG10078 in resisting cell damage

A long-chain non-coding and cell-damaging technology, applied in the field of molecular biology, can solve the problems of low lncRNA expression abundance, unconfirmed and undiscovered lncRNA, and achieve the effect of wide application value and resistance to damage

Active Publication Date: 2019-11-29
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, compared with protein-coding mRNA, the expression abundance of lncRNA is very low, and there are still a large number of lncRNAs that have not been confirmed and discovered.

Method used

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  • Rat long-chain non-coding lncRNA-lncMSTRG10078 and application of rat long-chain non-coding lncRNA-lncMSTRG10078 in resisting cell damage
  • Rat long-chain non-coding lncRNA-lncMSTRG10078 and application of rat long-chain non-coding lncRNA-lncMSTRG10078 in resisting cell damage
  • Rat long-chain non-coding lncRNA-lncMSTRG10078 and application of rat long-chain non-coding lncRNA-lncMSTRG10078 in resisting cell damage

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Embodiment 1 lncRNA-lncMSTRG10078 full-length sequence amplification

[0045] 1. Materials and Reagents

[0046] 1.1 Materials

[0047] The cell line used in this experiment is rat pituitary tumor cells (GH3 cells).

[0048] 1.2 Reagents

[0049] Phanta Max Super-Fidelity DNA Polymerase, Cat. No. P515, purchased from Nanjing Novizyme Biotechnology Co., Ltd.; HiScript II 1st Strand cDNA Synthesis Kit (+gDNA wiper), Cat. No. R212-01, bought from Nanjing Novozyme Biotech Co., Ltd. ; FastPure Gel DNA Extraction Mini Kit, item number DC301, purchased from Nanjing Nuoweizan Biotechnology Co., Ltd.; Mighty TA-cloning Reagent Set for Product No. 6019 was purchased from Bio-Technology (Beijing) Co., Ltd. (takara China); primers were synthesized by Nanjing GenScript Biotechnology Co., Ltd.

[0050] 2. Experimental method

[0051] 2.1 Extraction of total RNA

[0052] Total RNA was isolated from each GH3 cell sample in strict accordance with Sangon Bioengineering (Shanghai) ...

Embodiment 2

[0066] The full length of lncRNA-lncMSTRG10078 was identified using 5' and 3' RACE technology, and its full-length sequence was finally obtained as 1964bp, as shown in SEQ ID NO.1. Using UCSC and NCBI for sequence comparison, the results showed that lncRNA-lncMSTRG10078 was mainly located on chromosome 1 of rats (antisense strand, from 153859895-153861846) ( figure 1 ). Apply NCBI ORF-Finder and CPC, figure 2 It can be concluded that lncRNA-lncMSTRG10078 does not have protein coding ability, and the above results prove that lncRNA-lncMSTRG10078 is an lncRNA with a full length of 1964bp and no protein coding ability. Cloning and Analysis of Example 2 lncRNA-lncMSTRG10078 Sequence

[0067] 1. Reagents and carriers

[0068] Phanta Max Super-Fidelity DNA Polymerase, Cat. No. P505, purchased from Nanjing Novizyme Biotechnology Co., Ltd.; HiScript II 1st Strand cDNA Synthesis Kit (+gDNA wiper), Cat. No. R212-01, bought from Nanjing Novozyme Biotech Co., Ltd. ; FastPure Gel DNA ...

Embodiment 3

[0083] Example 3: Functional verification of lncRNA-lncMSTRG10078

[0084] 1. GH3 cell culture

[0085] GH3 cells were recovered in 5 mL DMEM complete medium (10% FBS + 90% DMEM + 1 mL double antibody + 1 mL glutamine) and placed in an incubator with 95% relative humidity at 37°C and 5% CO2 nourish. When the growth state of the cells reaches 70% to 90%, the trypsin-EDTA digestion method is used to pass passage at a ratio of 1:2, and pass passage once every 1-2 days. Cell cryopreservation adopts cell cryopreservation medium (90% FBS+10% DMSO) to carry out cryopreservation.

[0086] 2. Exploration of GH3 cell transfection conditions

[0087] The day before transfection, trypsinize the cells and count (it is advisable to make the density reach about 90% before transfection after about 24 hours), and plate the cells in 1 mL of normal growth medium containing serum and without antibiotics; Dilute 1:1, 1:1.5, 1:2, 1:2.5, 1:3 ExFect Transfection Reagent with 50 μL of serum-free O...

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Abstract

The invention discloses a rat long-chain non-coding lncRNA-lncMSTRG10078 and an application of the rat long-chain non-coding lncRNA-lncMSTRG10078 in resisting cell damage. The sequence of the rat long-chain non-coding lncRNA-lncMSTRG10078 is shown as SEQ ID NO.1 or has homology with more than 190% of SEQ ID NO.1; according to the invention, objective existence of the sequence is proved through PCR; a full-length transcript of the sequence can be cloned; a pcDNA 3.0-lncMSTRG10078 overexpression vector is constructed, GH3 cells are transfected, and results show that overexpression of lncMSTRG10078 can significantly regulate mitochondrial respiratory chain subunits, inflammation, apoptosis and metabolic changes so as to resist cell damage; flow results can show that overexpression of lncMSTRG10078 can significantly inhibit the generation of reactive oxygen species (ROS) and apoptosis, and further prove that the sequence can enhance the effect of cells on resisting cell damage.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a rat long-chain non-coding lncRNA-lncMSTRG10078 and its application in resisting cell damage. Background technique [0002] LncRNA is a class of RNA molecules with a transcript length of more than 200nt, which lacks a specific and complete open reading frame and does not have the ability to encode proteins. According to the relative position of lncRNA in the genome, it is divided into five categories: sense lncRNA, antisense lncRNA, bidirectional lncRNA, intergenic lncRNA, and intragenic lncRNA. According to their functions, lncRNAs can be divided into four types: signal molecules, decoy molecules, guide molecules and backbone molecules. These lncRNAs were once considered to be "junk sequences" accumulated in the evolution process, which are by-products of RNA polymerase II transcription and have no biological function, so they have not been given enough attention. W...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/85A61K31/7105
CPCA61K31/7105C12N15/113C12N15/85C12N2310/10
Inventor 王旭袁宗辉陆启荣潘源虎陈冬梅陶燕飞刘振利彭大鹏程古月郝海红谢书宇
Owner HUAZHONG AGRI UNIV
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