CN110511933B - Rat long-chain non-coding lncRNA-lncMSTRG10078 and application thereof in resisting cell injury - Google Patents

Abstract

The invention discloses a rat long-chain non-coding lncRNA-lncMSTRG10078 and application thereof in resisting cell injury, wherein the sequence is shown as SEQ ID NO.1 or has more than 190 percent of sequence homology with SEQ ID NO. 190; the objective existence of the sequence is verified through PCR; cloning the sequence full-length transcript, constructing pcDNA3.0-lncMSTRG10078 overexpression vector, transfecting GH3 cells, and the result shows that the overexpression of lncMSTRG10078 can obviously regulate and control mitochondrial respiratory chain subunit, inflammation, apoptosis and metabolic change, thereby resisting the cell injury effect; as can be seen from flow results, the over-expression of lncMSTRG10078 can obviously inhibit the generation of Reactive Oxygen Species (ROS) and the occurrence of apoptosis, and further proves that the sequence can enhance the effect of cells on resisting cell damage.

Description

Rat long-chain non-coding lncRNA-lncMSTRG10078 and application thereof in resisting cell injury

Technical Field

The invention relates to the technical field of molecular biology, in particular to a rat long-chain non-coding lncRNA-lncMSTRG10078 and application thereof in resisting cell injury.

Background

lncRNA is a class of RNA molecules with transcripts over 200nt in length that lack a specific complete open reading frame and do not have the ability to encode proteins. The lncRNA is divided into 5 classes of sense lncRNA, antisense lncRNA, bidirectional lncRNA, intergenic lncRNA and intragenic lncRNA according to the relative position of the lncRNA in a genome, and the position relationship is very helpful for predicting the biological function of the lncRNA. lncRNA is divided into 4 types of molecules such as signal molecules, decoy molecules, guide molecules and framework molecules according to functions. These lncRNAs were thought to be "garbage sequences" accumulated during evolution, are byproducts of RNA polymerase II transcription, are biologically nonfunctional, and therefore do not give sufficient attention. With the continuous development of sequencing technology, people gradually untie the mysterious veil of lncRNA, and more evidences show that lncRNA is a transcript with multiple functions. Recent studies show that lncRNA is widely involved in a plurality of important biological processes such as cell differentiation and development, chromosome silencing, genome imprinting, chromatin modification, transcriptional activation, transcriptional interference and the like.

Cell damage is mainly reflected in inflammation, apoptosis, metabolic changes, impairment of mitochondrial function, and the like. ROS are products of cellular metabolism, act on mitochondria, and cause damage to mitochondria; due to the fact that cells cannot metabolize and process ROS in time, ROS are accumulated in a large amount in the cells, cell dysfunction is caused, cell damage is caused, and cell apoptosis and CYP450 metabolic enzyme change are caused finally. However, lncRNA is expressed in low abundance compared to protein-encoding mRNA, and a large amount of lncRNA has not been confirmed and found. Therefore, research finds and identifies novel lncRNA, and has important significance in research on resisting cell damage.

Disclosure of Invention

Aiming at the prior art, the invention aims to provide a rat long-chain non-coding RNA-lncMSTRG10078 and an action mechanism thereof in resisting cell injury, and further apply the long-chain non-coding RNA to resist the cell injury.

In order to achieve the purpose, the invention provides the following scheme:

as a first aspect of the present invention:

the invention provides a rat long-chain non-coding lncRNA, which is lncMSTRG10078 and has a sequence shown in SEQ ID NO. 1.

Further, the rat long-chain non-coding lncRNA sequence also comprises a mutant, an allele or a derivative generated by adding, substituting, inserting or deleting one or more nucleotides in the sequence shown in SEQ ID NO. 1.

As a second aspect of the present invention:

the invention provides a eukaryotic overexpression vector which contains the rat long-chain non-coding lncRNA.

As a third aspect of the present invention:

the invention provides application of the rat long-chain non-coding lncRNA in resisting cell injury.

And the application of the eukaryotic overexpression vector in resisting cell injury.

As a fourth aspect of the present invention:

the invention provides a method for resisting cell damage, which comprises the following steps:

(1) constructing the eukaryotic over-expression vector of the rat long-chain non-coding RNA;

(2) the eukaryotic over-expression vector is transfected into cells, so that the expression amount of the rat long-chain non-coding RNA is increased.

The invention discloses the following technical effects:

the invention firstly separates a rat long-chain non-coding RNA lnc-MSTRG10078 related to resisting cell injury from a rat. The lnc-MSTRG10078 can be used as a gene resource or developed into a nucleic acid medicament to be used in agricultural production, livestock and poultry breeding and human disease treatment, is used for resisting the damage of exogenous medicaments or poisons to organisms, and has wide application value.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.

FIG. 1 is a diagram of the genomic position and structure of lncRNA-lncMSTRG10078 in rat;

FIG. 2 is a graph showing the results of analysis of the coding capacity of lncRNA-lncMSTRG10078, where A is the result of NCBI ORF-Finder prediction and B is the result of CPC website prediction;

FIG. 3 is a graph showing the amplification result of clone lncRNA-lncMSTRG10078, wherein M represents a DNA marker, the DNA molecular weight standard is DL5000, lanes 1-4 represent the amplified band of lncMSTRG10078, and the size is 1964 bp;

FIG. 4 shows the double restriction of Quickcut HindIII and Quickcut XhoI to identify the over-expression vector pcDNA3.0-lncMSTRG10078, the plasmid size of the over-expression vector is 7334bp, and the used DNA marker is DL15000 bp;

FIG. 5 is a chart of pcDNA3.0-EGFP transfection conditions;

fig. 6 is a graph of lncmsrg 10078 overexpression verification results, expressed as Mean ± SD (n ═ 3), indicating that p <0.05, which are significantly different from those in the group; p <0.01, significant differential from the group;

fig. 7 is a lncmsrg 10078 map of regulated mitochondrial-associated gene expression, with results expressed as Mean ± SD (n ═ 3) · indicating p <0.05, significantly different from those in the group; p <0.01, significant differential from the group;

fig. 8 is a graph of lntmstrg 10078 regulating expression of inflammation-associated genes, with results expressed as Mean ± SD (n ═ 3) · indicating that p <0.05, significantly different from those in the group; p <0.01, significant differential from the group;

fig. 9 is a graph of lntmstrg 10078 regulating expression of apoptosis-related genes, with results expressed as Mean ± SD (n ═ 3) · indicating that p <0.05, significantly different from those in the group; p <0.01, significant differential from the group;

fig. 10 is a graph of lncmsrg 10078 expression of genes involved in regulating metabolism, expressed as Mean ± SD (n ═ 3), indicating that p is <0.05, with significant differences compared to the group; p <0.01, significant differential from the group;

FIG. 11 is a graph of the effect of lncMSTRG10078 on ROS regulation;

figure 12 is a graph of the effect of lncMSTRG10078 on the regulation of apoptosis.

Detailed Description

It is to be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure, as all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

As described in the background section, lncRNA is involved in various biological functions of the body, but research on lncRNA associated with cellular injury compared to mRNA is insufficient. Therefore, the invention aims to provide a long-chain non-coding RNA related to rat cell injury, and the long-chain non-coding RNA is applied to resisting cell injury.

The invention obtains long-chain non-coding RNA lncRNA-lncMSTRG10078 by using a cDNA terminal rapid amplification technology (RACE). The comparison analysis of UCSC and NCBI shows that lncRNA-lncMSTRG10078 is positioned on chromosome 1 of rat genome, the length is 1964bp, the nucleotide sequence is shown in SEQ ID NO.1, and the structure is shown in FIG. 1.

Due to the specificity of the nucleotide sequence, any variant of the polynucleotide shown in SEQ ID NO.1 is within the protection scope of the present invention as long as it has more than 90% homology with the nucleotide and has the same function. A variant of a polynucleotide refers to a polynucleotide sequence having one or more nucleotide changes, including substitution, deletion and insertion variants.

The term "homologous" is intended to mean mainly homologous in sequence, i.e.to indicate that two or more RNA or DNA sequences have identical ancestry. Homologous sequences generally have similar functions. The homology between RNA and DNA is often determined by the sequence similarity, which is used to describe the ratio of identical DNA bases or amino acid residues between the test sequence and the target sequence during the alignment process. Generally, when the degree of similarity is higher than 50%, it is often presumed that the detection sequence and the target sequence may be homologous sequences; when the degree of similarity is less than 20%, it is difficult to determine whether or not they have homology.

The encoding capacity of lncMSTRG10078 is analyzed by an encoding capacity prediction website, and the lncMSTRG10078 is shown to have no protein encoding capacity.

The invention also uses homologous recombination method to clone lncMSTRG10078 sequence, constructs over-expression vector, cuts the over-expression vector pcDNA3.0 by HindIII and XhoI enzyme, recovers linearized over-expression vector pcDNA3.0 by glue and connects with target segment, tests and identifies, constructs eukaryotic expression vector. The liposome transfection reagent is used for respectively transfecting an empty vector pcDNA3.0 and a target vector pcDNA3.0-lncMSTRG10078 to GH3 cells, and the result of fluorescent quantitative PCR analysis shows that the expression quantity of the related target gene of lncMSTRG10078 can obviously resist the cell damage effect when the target vector pcDNA3.0-lncMSTRG10078 is transfected compared with a transfected empty vector group.

The generation of ROS and apoptosis is detected by a flow cytometer in cells transfected with an empty vector pcDNA3.0 and a target vector pcDNA3.0-lncMSTRG10078 to GH3 respectively by using a liposome transfection reagent, and the result shows that compared with a transfected empty vector group, the generation of ROS and apoptosis can be remarkably reduced by transfecting the target vector pcDNA3.0-lncMSTRG10078, and the cell damage effect is further resisted.

In conclusion, the eukaryotic overexpression vector is utilized to increase the expression level of rat lncRNA-lncMSTRG10078, so that the damage effect of cells can be obviously resisted.

In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the present application will be described in detail with reference to specific embodiments.

The test materials used in the examples of the present invention are all conventional in the art and commercially available. The experimental procedures, for which no detailed conditions are indicated, were carried out according to the usual experimental procedures or according to the instructions recommended by the supplier.

Example 1 amplification of the full Length of lncRNA-lncMSTRG10078

1. Materials and reagents

1.1 materials

The cell line used in this assay was rat pituitary tumour cells (GH3 cells).

1.2 reagents

Phanta Max Super-Fidelity DNA Polymerase, cat number P515, available from Nanjing Nodezam Biotech Co., Ltd; HiScript II1st Strand cDNA Synthesis Kit (+ gDNA wiper), cat # R212-01, purchased from Biotech, Inc., of Nakyo Weizan, Nanjing; FastPure Gel DNA Extraction Mini Kit, cat # DC301, available from Nanjing Novowed Biotech, Inc.; light TA-cloning Reagent Set for

Cat # 6019, available from baoriri physician science, inc (tokara, china); the primers were synthesized by Nanjing Kingsrei Biotechnology, Inc.

2. Experimental methods

2.1 extraction of Total RNA

Total RNA was isolated from each GH3 cell sample strictly according to the preparative total RNA extraction and purification kit (B518651) from Biotechnology engineering (Shanghai) Ltd. The RNA concentration was calculated by measuring OD260 and OD280 of the total RNA samples extracted using Quawell Q3000. 1 μ g of the total RNA sample was subjected to 1.0% agarose gel electrophoresis to determine the integrity of the total RNA.

2.2 primer design

According to the known nucleotide sequence of part lncRNA-lncMSTRG10078, primers are designed by referring to a primer calculation tool of Nanjing Jinsley Co., Ltd, and the primer sequences are as follows:

2.3 reverse transcription PCR

The reverse transcription primers used in this experiment were QT and GSP-RT, the primers were diluted according to the order of primer Synthesis, 1. mu.L of 3 'RACE and 5' RACE was used per 20. mu.L of reverse transcription system, and 80. mu.L of cDNA was synthesized completely according to the Novozan HiScript II1st Strand cDNA Synthesis Kit.

RNaseH treatment: RNA was removed from the DNA-RNA hybrid strand, and 2. mu.L of Reaction Buffer (10X), 17.8. mu.L of DEPC water, and 0.2. mu.L of RNase H (5U/. mu.L) were sequentially added to each 20. mu.L Reaction system in which the first strand had been synthesized on an ice bath, gently mixed (either by pipetting or by Vortex) and then centrifuged to precipitate the liquid. After incubation at 37 ℃ for 1h, 2.5. mu.L of 0.5M EDTA (pH8.0) was added thereto and mixed well to terminate the reaction, and the synthesized double-stranded cDNA was subsequently purified by ethanol precipitation.

Description of the drawings: in other cases, the conditions for removing RNA from the DNA-RNA hybrid strand can be performed by referring to the above conditions. The pH range for RNase H reaction may be about 7.5-8.3.

2.43 'RACE and 5' RACE

3 'RACE and 5' RACE were performed with reference to the classical RACE method in the PCR technical Experimental guidelines (original 2 nd edition).

2.5 analysis of the Structure and coding Capacity of IncRNA-IncMSTRG 10078

The UCSC and NCBI websites are used for analyzing the structure of the full-length sequence of the lncRNA-lncMSTRG10078, and the NCBI ORF-Finder and CPC websites are used for analyzing the coding capacity of the lncRNA-lncMSTRG 10078.

3. Results of the experiment

The 5 'and 3' RACE technology is applied to identify the full length of lncRNA-lncMSTRG10078, and the finally obtained full length sequence is 1964bp and is shown as SEQ ID NO. 1. The results of sequence alignment using UCSC and NCBI showed that lncRNA-lncMSTRG10078 is mainly located on chromosome 1 (antisense strand, from 153859895-153861846) in rat (FIG. 1). Using NCBI ORF-Finder and CPC, it can be seen from FIG. 2 that IncRNA-IncMSTRG 10078 has no protein-coding ability, and the above results demonstrate that IncRNA-IncMSTRG 10078 is an IncRNA having a full length of 1964bp and no protein-coding ability. Example 2 cloning and analysis of the IncRNA-IncMSTRG 10078 sequence

1. Reagents and carriers

Phanta Max Super-Fidelity DNA Polymerase, cat # P505, available from Nykano Zanza Biotech, Inc.; HiScript II1st Strand cDNA Synthesis Kit (+ gDNA wiper), cat # R212-01, purchased from Biotech, Inc., of Nakyo Weizan, Nanjing; FastPure Gel DNA Extraction Mini Kit, cat # DC301, available from Nanjing Novowed Biotech, Inc.; clonexpress Ultra One Step Cloning Kit, cat # C115, available from Nyvamin technologies, Inc., Nanjing; light TA-cloning Reagent Set for

Cat # 6019, available from baoriri physician science, inc (tokara, china); quickcut HindIII and Quickcut XhoI, available from Takara, Inc. (Beijing) of Baozi medical technology, Inc.; gel recovery (D2500-02) kit and endotoxin-removing plasmid extraction kit (D6950-01) were purchased from OMEGA; the primers were synthesized by Nanjing Kingsrei Biotechnology, Inc.

2. Method of producing a composite material

2.1 extraction of Total RNA

The experimental procedure was the same as 2.1 total RNA extraction in example 1.

2.2 primer design

According to the lncRNA-lncMSTRG10078 nucleotide sequence, as shown in SEQ ID NO.1, the lncRNA-lncMSTRG10078 amplification primer is designed by applying the design principle of homologous recombinase, which comprises the following specific steps:

lncMST F actatagggagacccaagcttTTCCCAATGGATTCAAGGAGC；lncMST R ccctctagatgcatgctcgagTTTTTTTTTTTTTGACTGTTTCAATCTTTTAAACAAGATT

2.3 first Strand cDNA Synthesis reaction

The reverse transcription reaction is divided into three steps to extract total RNA: (<5 μ g) as a template, first, the RNA template was denatured, and oligo (dT) was prepared as a reaction solution in an RNase-free centrifuge tube₂₃VN (50. mu.M), addition of RNA and RNase-free ddH₂After O, the volume is reduced to 12 mu L, the reaction solution is heated at 65 ℃ for 5min, and the reaction solution is quickly heatedQuickly quench on ice and stand on ice for 2 min. After denaturing the reaction solution, 4 XgDNA wiper Mix 4. mu.L was added thereto, and the mixture was gently pipetted and mixed. Genomic DNA was removed at 42 ℃ for 2 min. And continuously adding 2 mu L of 10 xRT Mix and 2 mu L of HiScript II Enzyme Mix into the mixed solution in the previous step, gently blowing and stirring the mixture by a pipette, and performing reverse transcription reaction at 50 ℃ for 45min and 85 ℃ for 2min to obtain first strand cDNA.

2.4 IncRNA-IncMSTRG 10078 transcript PCR amplification

The PCR reaction was carried out on a PCR instrument using the high Fidelity enzyme Phanta Max Super-Fidelity DNA Polymerase kit, and the reaction system was as follows: 2 × Phanta Max Buffer 25 μ L, dNTP Mix (10mM each)1 μ L, lncMSTRG10078 specific upstream and downstream primers each 2 μ L, template DNA 2 μ L, Phanta Max Super-Fidelity DNA Polymerase 1 μ L, ddH₂O to 50. mu.L. The reaction procedure is pre-denaturation at 95 ℃ for 3 min; denaturation 95 ℃ for 15sec, annealing 55 ℃ for 15sec, elongation 72 ℃ for 120sec, and circulation 35 times; then extended for 5min at 72 ℃. The obtained PCR products were subjected to agarose electrophoresis detection, photographed using a gel imager, the resulting fragment size analyzed (FIG. 3) and the correct single-purpose band excised.

2.5 construction of IncRNA-IncMSTRG 10078 overexpression vector

The over-expression vector pcDNA3.0 is subjected to double digestion by Quickcut Hind III and Quickcut XhoI, and a correct single band is cut after agarose gel electrophoresis. And (3) carrying out gel recovery on the target gene band in the step 2.4 and the band subjected to double enzyme digestion of the overexpression vector, wherein the gel recovery step is carried out according to an OMEGA gel recovery kit. The gel recovered DNA solution was ligated with 2 XCloneexpress Mix in the following system: the linearized vector was 2. mu.L of 3. mu. L, lncMSTRG10078 mesh fragment and 2 XCloneexpress Mix 5. mu.L, gently mixed by pipetting, the reaction solution was collected to the bottom of the eye by brief centrifugation, the mixture was placed in a PCR apparatus at 50 ℃ for 15min, cooled to 4 ℃ or immediately placed on ice and cooled. The ligation products were transformed into Trans-T1 phase resist chemically competent cells, 50. mu.L of the competent cells thawed on ice bath were added with 5. mu.L of the ligation product, gently mixed, placed in ice bath for 30 minutes, heat-shocked in a water bath at 42 ℃ for 30 seconds, and then the tubes were quickly transferred into the ice bath for 2 minutes without shaking the centrifuge tubes. mu.L of sterile LB medium without antibiotics was added to each tube, mixed well and incubated at 37 ℃ for 1 hour at 200rpm to resuscitate the bacteria. After centrifugation at 5000rpm for 1min, the supernatant was discarded to the remaining 100. mu.L, and the resuspended cell pellet was added to LB agar medium containing ampicillin to spread the cells evenly. Placing the plate at 37 ℃ until the liquid is absorbed, inverting the plate, culturing at 37 ℃ overnight, picking out monoclonal shake bacteria for 12h, extracting plasmids, performing double enzyme digestion identification by using Quickcut HindIII and Quickcut XhoI (figure 4), and sending the correctly identified monoclonal vector to the sequencing identification of Biotechnology engineering (Shanghai) GmbH.

3. Results of the experiment

The fragment length of the obtained positive clone is 1964bp and the sequence is correct by agarose electrophoresis detection and sequencing result analysis, which confirms the real existence of lncRNA-lncMSTRG 10078.

Example 3: functional verification of lncRNA-lncMSTRG10078

GH3 cell culture

GH3 cells were recovered in DMEM complete medium (10% FBS + 90% DMEM +1mL diabody +1mL glutamine) containing 5mL and cultured in an incubator at 95% relative humidity, 37 ℃ and containing 5% CO 2. When the cell growth state reaches 70% -90%, adopting a trypsin-EDTA digestion method according to the ratio of 1:2 ratio passage, once every 1-2 days. Cell cryopreservation was performed using cell cryopreservation solution (90% FBS + 10% DMSO).

2. GH3 cell transfection conditions

One day before transfection, cells were trypsinized and counted (to achieve a pre-transfection density of around 90% after about 24 hours), and the cells were plated in 1mL of serum-free, antibiotic-free medium for normal growth; diluting 1:1, 1:1.5, 1:2,1:2.5,1:3 ExFect Transfection Reagent with 50 μ L of non-serum Opti-MEM medium, standing and activating for 5 min; for each well of cells, 1. mu.g of plasmid was diluted with 50. mu.L of serum-free Opti-MEM medium, gently mixed and then left at room temperature for 5 min; mixing the diluted ExFect Transfection Reagent with DNA, gently mixing uniformly, and standing at room temperature for 20min to form a DNA/ExFect Transfection Reagent mixture; treating the cells while standing for 20min, and then adding 900. mu.L of opti-MEM; adding the mixture of the ExFect Transfection Reagent mixture to each well, gently shaking the cell culture plate back and forth, and marking; after 6h, the medium was changed to non-antibody complete medium and fluorescence was recorded at 12, 24, 36 and 48 hours post-transfection to determine the optimal transfection concentration and time (FIG. 5).

3. lncRNA-lncMSTRG10078 transfection

The optimal transfection concentration and time were determined according to 2 (1: 2.5, transfection 24 h). One day before transfection, cells were trypsinized and counted (to achieve a pre-transfection density of around 90% after about 24 hours), and the cells were plated in 1mL of serum-free, antibiotic-free medium for normal growth; according to the DNA: the Transfection Reagent was activated by standing for 5min with 2.5. mu.L of ExFect Transfection Reagent diluted in 50. mu.L of serum-free Opti-MEM medium at a ratio of 1: 2.5; for each well of cells, 1. mu.g of plasmid was diluted with 50. mu.L of serum-free Opti-MEM medium, gently mixed and then left at room temperature for 5 min; mixing the diluted ExFect Transfection Reagent with DNA, gently mixing uniformly, and standing at room temperature for 20min to form a DNA/ExFect Transfection Reagent mixture; treating the cells while standing for 20min, and then adding 900. mu.L of opti-MEM; the extract Transfection Reagent mixture was added to each well, the cell culture plate was gently shaken back and forth, labeled, replaced with non-antibody complete medium after 6h, and harvested 24h after Transfection. Fluorescent quantitative PCR detection of lncRNA-lncMSTRG10078 related functional gene

4.1 Total RNA extraction

The experimental procedure was the same as 2.1 total RNA extraction in example 1.

4.2 primer design

According to the nucleotide sequence of a related target gene, a qRT-PCR primer is designed through NCBI primer Blast, the length of a product is 70-300bp, and beta-actin with stable expression quantity is used as an internal reference gene. The primers were synthesized by Nanjing Kingsrei Biotechnology, Inc.

The primer sequences are as follows:

4.3 reverse transcription reaction

The reverse transcription reaction is divided into two steps, 1 mug of extracted total RNA is taken as a template, firstly, the genome DNA is removed, and reaction solution oligo (dT) is prepared in an RNase-free centrifuge tube₂₃VN (50. mu.M) 1. mu.L, Random hexamers (50 ng/. mu.L) 1. mu.L, 4 XgDNA wiper Mix 4. mu.L, addition of RNA and RNase-free ddH₂And O, then the volume is 16 mu L, the mixture is lightly blown and beaten by a pipette and is uniformly mixed, the mixture is heated at 42 ℃ for 2min and is rapidly placed on ice for quenching, 10 xRT Mix 2 mu L and HiScript II Enzyme Mix 2 mu L are continuously added into the reaction solution, and after the mixture is lightly blown and beaten by the pipette and is uniformly mixed, reverse transcription reaction is carried out at 50 ℃ for 15min and 85 ℃ for 2min to obtain the first strand cDNA.

4.4 Quantitative real-time(RT)PCR

PCR amplification was performed on a CFX96 Real-Time PCR Detection System Real-Time fluorescent quantitative PCR instrument using the ChamQ Universal SYBR qPCR Master Mix kit, and the qRT-PCR reaction System was prepared on ice as follows:

after the reaction mixture is slightly centrifuged, PCR amplification is carried out on a CFX96 Real-Time PCR Detection System Real-Time fluorescent quantitative PCR instrument, and the reaction program is as follows:

the first step is 30s at 95 ℃;

the second step is that 10s at 95 ℃; tm value corresponding to 30s at, 40 cycles in total;

the third step is that 10s at 95 ℃; 65 ℃ to 95 ℃ with 0.5 ℃ increments. The amplification curve and the lysis curve of each gene were analyzed. By Ct comparison method and using beta-actin as reference gene, namely 2^-ΔΔCtTo measure the difference in transcription level. The experiment was repeated 3 times.

5. Results of the experiment

The expression of lncMSTRG10078 in GH3 cells overexpressing pcDNA3.0 and pcDNA3.0-lncMSTRG10078 is shown in FIG. 6, and thus it can be seen that lncMSTRG10078 is successfully overexpressed. As can be seen from fig. 7, 8, 9 and 10, lntmsrg 10078 can significantly inhibit and control changes of mitochondrial respiratory chain-related subunits, significantly inhibit inflammation, significantly inhibit apoptosis, and significantly regulate changes of CYP450 enzymes related to mitochondrial metabolism. Experimental results show that the lncMSTRG10078 has an important function in GH3 cells over-expressing pcDNA3.0 and pcDNA3.0-lncMSTRG10078, can regulate and control the generation of mitochondrial respiratory chain subunits, inflammation and apoptosis and the angle of metabolic change, and further resists the action of cell injury.

Example 4 regulation of ROS and apoptosis by lncRNA-lncMSTRG10078

1.GH3 cell culture

GH3 cell culture conditions were the same as 1 in example 3.

2. lncRNA-lncMSTRG10078 transfection

lncRNA-lncMSTRG10078 was transfected as in 3 of example 3.

3. ROS assay

GH3 cells were cultured, inoculated into 12-well plates, transfected with pcDNA3.0 and pcDNA3.0-lncMSTRG 1007824 h, and then added to a medium containing 10. mu.M DCFH-DA. Cells were incubated for 1h at 37 ℃. Digesting the cells with 0.25% pancreatin for 30s, adding complete medium to terminate digestion, making a cell suspension, centrifuging for 5 minutes at 1000g, washing with PBS for 1-2 times, centrifuging to collect cell precipitates, resuspending the cells with 500 μ LPBS, and performing ROS measurement with Beckman FC 500.

4. Apoptosis assay

GH3 cell culture, inoculating to 12-well plate, transfecting pcDNA3.0 and pcDNA3.0-lncMSTRG 1007824 h respectively, digesting the cells for 30s by 0.25% pancreatin without EDTA, adding complete culture medium to stop digestion to prepare cell suspension, centrifuging at 2000rpm for 5 minutes at room temperature, resuspending the cells once by precooled PBS, centrifuging at 2000rpm for 5 minutes, washing and washing the cells, adding 300 mu L of 1 XBinding Buffer suspension cells, adding 5 mu L of Annexin V-FITC, mixing uniformly, keeping out of the sun, and incubating at room temperature for 15 minutes; 5 mu L of PI staining is added before the machine is operated for 5 minutes, 200 mu L of 1 XBinding Buffer is added before the machine is operated, and the cell apoptosis is measured by Beckman FC 500.

5. Results of the experiment

From the flow results of fig. 11 and fig. 12, it can be found that pcdna3.0 and pcdna3.0-lncmtrg 10078 are overexpressed in GH3 cells, and lncmtrg 10078 can significantly inhibit the generation of ROS and significantly inhibit the apoptosis rate of cells, further indicating that lncmtrg 10078 has important role and function in resisting cell damage.

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.

Sequence listing

<110> university of agriculture in Huazhong

<120> rat long-chain non-coding lncRNA-lncMSTRG10078 and application thereof in resisting cell injury

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1964

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

ttcccaatgg attcaaggag ctagcccccg cccctcgtcc gaggccacct gctccgtgct 60

tggttgcgat gttcaccccc gcagccaaca aagcgaaagc aagcccagaa agcagatccc 120

gtgagggtga cagcctgtgc tggcctccat acagagaata ctctccgagg ggtccctgca 180

ccctcagtac ctgaggttgg ccttgtcttc tccacttacc tgcctaactg gagaagctca 240

agtctgtgag aagctcaagt ctgtgcagcc ccactgcagg caggtaggga aagacgctac 300

tgtcccctgt cacactctgg ccaaaggcag caccgcacca ctcaggcagt acgagggact 360

gctgtgttca gtaaaatcct tgccttgcag gaccagactt cccttgagga tacgcgtggg 420

ggctcttcgt atcaagatgt aacaagatga agctcacttt cctcccacat gagggctttt 480

gtgcttttgt accttggcac tctctatcca gaggacttga ggatcatcat gctccctgcc 540

tcttgagcaa ggctctgagc aaggatgctg tcctgtgaag taaccattag ccacatgtag 600

ctgtaatact ctaaataaaa tacaatttaa aatccatttc cccagtccca gcaagcagtt 660

ctttttaaag cagttgggag catgtgtgag tgcatatagg tacagaacac ttccaacttt 720

gcagaacgtt ctcttgtctt gatctgagtc tcccacttcc tagggaggtt tctaagaacc 780

ccatgaagcc agtcatagat gttagggtca caccttaatc cctgaattta aatgtatact 840

tcctctcagt attcaagcga ttccttctga aggaaataca tctcactgtg tatgggataa 900

cacccaagtc gtcaggaggc cttggttaac cccagcctgg ttcctgacaa ggtctaatgc 960

ttgtcctact tcacactgtg ctgtccttga ggtctcccct cacttgcttc tccccatttc 1020

tccaccttac ttccctgcct ccacctcggg gcttttgtat ctgctaatgc ttttgcatgg 1080

atcctcacac tccaatcttc caaacaactg ccacatctca tctctgaggt ggcccctaat 1140

atgtcaactt gggggccttt attgtaggta ttcccttcag tctgaccatc actaggggga 1200

acataggaaa aaggtatccc ctatgtgtaa acgaacaagt cagttaccag ctcacagctc 1260

ttgtgcttta ttctctgcct cccccttaca ctacaggctt cactgggctt gggtctttac 1320

ctgcaaatcc atttgtcctc agggcttgac gtgtctttgt acaataaaaa catgtttggt 1380

gagggaaggt gagtggcttc cctgttgatc aggactatga gctctcagaa tggctgccag 1440

acagatggtg tggttctctc agactcactt tcaaaccgca tcccattctc tggtcccaac 1500

tgcaaggcag gcataatggt actgtcaggg tcctcctgcc tggcctcagg attccatctc 1560

ctcggaagtg atgggaatta gtgccttccc ctgaagcctc tgtcctttct gctccaattt 1620

ctagttctca gctagaatga tctatattca attgtactgg ataaaagaat gtcgattctc 1680

tttagatgat atttggtata taggtcacac ttcagagcag gccccatgcc ctggactaat 1740

tagctatctg ggcaacacag actgaattcc atggggtttg tttgtttgtt tgttcatttg 1800

ttttgagtca gagggcaaac ataaagttgg atgggtagga aggaggggag gacctgggaa 1860

cagctgggag aacaagatga acatgatcga ttatgtttta tgaaacctcc aatgaataaa 1920

taaaaatctt gtttaaaaga ttgaaacagt caaaaaaaaa aaaa 1964

Claims (4)

1. A rat long-chain non-coding lncRNA is lncMSTRG10078, and the nucleotide sequence of the lncRNA is shown in SEQ ID NO. 1.

2. A eukaryotic overexpression vector comprising the rat long non-coding incRNA of claim 1.

3. Use of a long non-coding incrna according to claim 1 for the preparation of a medicament against cell damage.

4. Use of a eukaryotic overexpression vector as claimed in claim 2 for the preparation of a medicament for combating cell damage.

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