CN108410874B - Intestinal tissue marker lncRNAH19 after severe burn and application thereof - Google Patents

Intestinal tissue marker lncRNAH19 after severe burn and application thereof Download PDF

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CN108410874B
CN108410874B CN201810210930.8A CN201810210930A CN108410874B CN 108410874 B CN108410874 B CN 108410874B CN 201810210930 A CN201810210930 A CN 201810210930A CN 108410874 B CN108410874 B CN 108410874B
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孙勇
邱伟
张盼
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Abstract

The invention relates to an intestinal tissue marker, in particular to an intestinal tissue marker lncRNA H19 after severe burn, a detection method and application thereof. IncRNA-H19 was most significantly differentially expressed in the intestine of mice with scald, and was upregulated to express IncRNA. The influence of the Western Blot on the IEC-6 autophagy level after infection with H19 overexpression virus is detected, the autophagy level of cells is obviously increased after the experimental group is infected with Ad-m-H19-GFP, and the result shows that H19 has the effect of improving the IEC-6 autophagy level.

Description

Intestinal tissue marker lncRNAH19 after severe burn and application thereof
Technical Field
The invention relates to an intestinal tissue marker, in particular to an intestinal tissue marker lncRNA H19 after severe burn, a detection method and application thereof.
Background
The intestinal tract is the organ with the largest surface area in the human body and is also the largest bacteria storage organ in the human body. The burn is a systemic disease, the serious burn can cause the damage of large-area skin and subcutaneous tissues, each organ has damage of different degrees, can secondarily cause the damage of multiple organs such as heart, lung, stomach, intestinal tract, kidney and the like, the main injury factors are tissue ischemia and hypoxia damage, ischemia-reperfusion damage, systemic and gastrointestinal tract serious infection and the like, wherein the intestinal tract damage is particularly serious, the intestinal tract structure is damaged and dysfunctions after the serious burn can cause the imbalance and displacement of intestinal flora, the body immunity abnormity and excessive inflammatory reaction, thereby aggravating the damage of the intestinal tract homeostasis, further causing the intestinal infection and the intestinal hypermetabolism and further aggravating the damage of other organs, therefore, the intestinal tract is one of central organs of burn (wound) injury stress, and is one of central organs which causes the intestinal infection and the systemic inflammatory response syndrome (systemic inflammatory response syndrome), SIRS), and even the major cause of Multiple Organ Dysfunction Syndrome (MODS). Therefore, how to protect the intestinal mucosal barrier and promote the repair of damaged mucosa is an urgent problem to be solved, and is one of the hot spots of the current basic research and clinical treatment, and is also one of the core problems of serious medical attention including burn.
The irritable reaction of the intestinal tract after burn causes damage to the intestinal tract barrier, a series of biological processes can also occur to repair the intestinal tract mucosa barrier, and various mechanisms for repairing and damaging the intestinal tract barrier function relate to the fields of microorganisms, immunity, molecular biology and the like, and are quite complex processes. Currently, there are several common methods for clinically protecting intestinal mucosa: enteral nutritional support; supplementing probiotics and prebiotics; selectively decontaminating the digestive tract; the anti-radical agent prevents or reduces peroxidation damage and the like. However, the above-mentioned conventional solutions often only provide partial relief and make it difficult to completely restore the intestinal mucosal barrier. Therefore, genes for promoting intestinal mucosa repair are further explored at the gene level, and a novel intestinal mucosa repair treatment strategy based on the signal pathways can be provided by fundamentally researching the signal pathways and molecular mechanisms for repairing the intestinal mucosa under the natural condition of the organism.
With the development of biochips and high throughput sequencing technologies, it has been found that about 70-90% of genes in the human genome are transcribed to produce RNA, but less than 2% of the genes are transcribed to mRNA and finally translated to protein and exert their biological functions in the form of protein, while more than 98% of the genes are not transcribed to protein-coding, so-called non-coding RNA (ncRNA), transcription products thereof. lncRNA is a non-coding RNA of greater than 200nt in length, lacking an Open Reading Frame (ORF), and having no protein-coding function. However, as the research on lncrnas is intensive, more and more researches show that lncrnas with higher transcription rate in cells have extremely complex and important biological functions compared with micrornas, and the lncrnas are not only widely expressed in various tissues, but also have diverse functions. The current research considers that the functional action mechanism mainly comprises the interference of downstream gene expression, the influence of the transcription of the coding protein gene, the regulation of the protein function and the like. Can regulate and control the expression of genes at a plurality of levels, including regulating and controlling the expression of genes at epigenetics, transcription and post-transcription levels, participates in a plurality of important regulation and control processes such as X chromosome silencing, genome imprinting, chromatin modification, transcription activation, transcription interference, intranuclear transportation and the like, and is closely related to the occurrence, development and prevention and treatment of human diseases. Many researches show that lncRNA can participate in the regulation and control of gastrointestinal diseases, the differential expression of lncRNA is related to tumors, inflammatory bowel diseases and the like, and lncRNA can simultaneously regulate and control the expression of a plurality of target genes mRNAs, and macroscopically regulates and controls a biological process and a related signal path in a repair process in intestinal tissues after severe burn, so that the aim of treating diseases is integrally achieved.
Disclosure of Invention
The invention finds that the autophagy level of the intestinal tract is increased after severe burn, and the increase of the autophagy level can promote the repair of the damage of the intestinal mucosa, but the fundamental regulation and control of the gene level are still blank. Therefore, the invention screens markers related to intestinal tissue repair after severe burn by making a burn model, extracts total RNA, and screens lncRNAs and mRNAs with significant differential expression. Compared with intestinal tissues of mice in a sham-scald control group, 24H after scald have 1384 genes which are significantly and differentially expressed in the intestinal tissues of the mice, wherein 571 genes are up-regulated (lncRNA: 109, mRNA: 462), 813 genes are down-regulated (lncRNA: 45, mRNA: 768), and lncRNA-H19 has the most significant differential expression in the intestinal tracts of the scald mice and is up-regulated to express lncRNA. H19 was significantly up-regulated in intestinal tissue of 20 severely scalded mice. The influence of the Western Blot on the autophagy level of rat crypt epithelial cells (IEC-6 cells) after infection of H19 overexpression viruses is detected, the autophagy level of the cells is obviously increased after infection of Ad-m-H19-GFP in an experimental group, and the difference has statistical significance (P is less than 0.05), so that H19 has the effect of improving the autophagy level of the IEC-6 cells.
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FIG. 1: comparative analysis of qRT-PCR detection result and chip detection result
FIG. 2: h19 expression comparison in intestinal tissues of control group and severely scalded mouse
FIG. 3: western Blot to test the influence of infection with H19 over-expressed virus on the level of IEC-6 autophagy
Detailed Description
The following describes embodiments of the present invention in further detail.
Example 1 screening for markers associated with intestinal tissue repair after severe burn injury
1. Sample collection
The clean adult 20-25g C57BL/6J mice were selected as the study subjects. Manufacturing a burn model: pentobarbital sodium 1% pentobarbital sodium (0.1ml/10g) is injected into abdominal cavity, 8% sodium sulfate on the back is used for depilation, the back is placed in 100 ℃ boiled water for 10s to cause 30% TBSA III degree scald, sodium lactate ringer injection (50ml/kg) is immediately injected into abdominal cavity for antishock treatment, iodophor is smeared on the back to resist infection, and gauze is covered for heat preservation. A false wound model: the back was placed in a 37 ℃ water bath for 10 s.
1. RNA extraction
1) 50-100mg of small intestine ileocecal tissue is taken, placed on ice, added with 1ml of Trizol, fully homogenized by using a homogenizer, transferred into a new 1.5ml ribozyme-free EP tube, and kept stand for 10min at room temperature to fully crack the tissue.
2)12000rpm, 4 degrees C10 min, remove the impurity precipitation, suction supernatant to new RNase-Free1.5ml EP tube.
3) Adding 0.2ml chloroform (chloroform), shaking for 15s, standing at room temperature for 3-5min to separate.
4)12000rpm、4℃15min。
5) Carefully sucking the upper aqueous phase by a pipette, transferring the upper aqueous phase into a 1.5ml ribozyme-free centrifuge tube, adding equal volume of precooled isopropanol into the upper aqueous phase, fully mixing the mixture evenly, and standing the mixture for 10min at room temperature.
6) Centrifuging at 12000rpm and 4 deg.C for 10min, allowing precipitate to be visible at the bottom of the tube, discarding the supernatant, washing residual isopropanol with 1ml of 75% ethanol, centrifuging at 12000rpm and 4 deg.C for 10min, and discarding the supernatant.
7) Drying in a ventilation opening for 5min, adding 25ul RNase-Free water to dissolve RNA fully, and keeping on ice for later use.
3. And (3) detecting the total RNA quality:
the optical density values of total RNA at OD230nm, OD260nm,0D280nm were determined using a spectrophotometer. When the total amount of RNA is more than or equal to 3ug, OD260/230 is more than or equal to 0.7, OD260/280 is more than or equal to 1.8, and RIN is more than or equal to 7.0, the RNA can be used for the next experiment.
2. Screening for differentially expressed lncrnas:
the difference of lncRNA expression profiles in 24h intestinal tissues after burn and in intestinal tissues of sham-scald mice was detected by using Agilent Mouse V28 x 60K chip of Huada Gene technology, Inc. Agilent's mouse genome-wide expression profiling chip, detecting both coding and non-coding RNAs, covering 27,122 Entrez Genes mRNAs and 4,578 lncRNAs. The sequence information used for this chip was derived from RefSeq Build 66, Ensemble Release 76, Unigene Build236, GenBank, RIKEN 3. A plurality of probes are designed for each sequence, so that the reliability of signals is improved.
3. Screening of differentially expressed genes:
normalizing the original data of the chip detection, and screening lncRNAs and mRNAs (log) with significant differential expression2I Fold Change | ≧ 1.0 and P < 0.05).
4. As a result:
the chip result shows that compared with intestinal tissues of mice in a sham-scald control group, 1384 genes which are significantly and differentially expressed in the intestinal tissues of the mice 24H after scald are shared, wherein 571 genes are up-regulated (lncRNA: 109, mRNA: 462), 813 genes are down-regulated (lncRNA: 45, mRNA: 768), and lncRNA-H19 is the most significantly and up-regulated to express lncRNA in the intestinal tracts of the mice in scald.
Example 2 verification of differential expression of related genes by qRT-PCR sequencing
1. QPCR validation of six lncrnas was performed using the chip analysis remaining RNA samples (burn group N3, control group N3).
2. Reverse transcription:
1) configuration of reverse transcription system:
a20 ul reaction system was used, 2ug total RNA was taken as RNA template for each sample, 2ul random, 2ul SuperPure dNTPs (2.5mM each), and RNase-Free ddH was supplemented2The volume of O is up to 14.5 ul. Heating at 70 deg.C for 5min, and rapidly cooling on ice for 2 min.
After the reaction solution is collected by brief centrifugation, the following components are added: 4ul of 5 XFirst-Strand Buffer (containing DTT), 0.5ul of RNase, 1ul (200U) of TIANCcript M-MLV.
2) Reverse transcription reaction conditions:
performing subsequent experiment or freezing preservation on the mixture at 25 ℃ for 10min, 42 ℃ for 50min and 95 ℃ for 5min on ice.
3) QPCR amplification:
primers were designed as follows, synthesized from Shanghai:
Figure BDA0001597211080000041
Figure BDA0001597211080000051
preparing 20ul PCR reaction system:
with RNase-Free ddH2O dilution of cDNA to 50 ul.
Adding 2 XSuperReal PreMix Plus 10ul, forward primer (10uM)0.6ul, reverse primer (10uM)0.6ul, cDNA template 2ul, 50 XROX Reference Dye 0.4ul, RNase-free ddH2O6.4 ul. Each operation was performed on ice, and 3 parallel channels were set for each gene in each sample to ensure the reliability of the results.
And (3) amplification procedure:
the setting procedure is as follows: 95 ℃ 15min, (95 ℃ 10s, 57 ℃ 32s, 72 ℃ 32s) x 40 cycles.
SYBR Green is used as a fluorescent marker, PCR reaction is carried out on an ABI7500 fluorescent real-time quantitative PCR instrument, the specificity of amplification is judged through a dissolution curve, and relative quantification is carried out by a delta-delta CT method.
3. As a result:
as shown in FIG. 1, compared with the control tissue, the expression of H19, NR-040769, Bvht and Dnm3os is up-regulated in the intestinal tract tissue of the burned mice, and the expression of Dleu-2 and 2010010A06Rik is down-regulated in the intestinal tract of the burned mice, and the difference has statistical significance (P is less than 0.05), and is consistent with the chip result, so that the reliability of the chip result is proved.
Example 3 qRT-PCR sequencing verified differential expression of the lncRNA-H19 gene:
1. large sample QPCR validation was performed on differential expression of the H19 gene. Ileocecal intestinal tissue after severe burns (N-20) and control (N-15) were selected according to the collection of the samples in (one).
2. RNA extraction step as in (I):
and (3) detecting the total RNA quality:
the ultraviolet spectrophotometer is used for determining the concentration and the purity of the total RNA:
2ul of total RNA sample was taken and added to 98ul of DEPC water, absorbance values at 230nm, 260nm and 280nm were measured, and the ratio OD260/OD280 was calculated, which was between 1.8 and 2.1 indicating better purity. OD260/OD230 > 2.0, and the closer to 2.5, the higher the RNA purity.
The sample RNA concentration (ug/ml) ═ a260 × 40ug/ml × dilution factor.
3. Reverse transcription: according to the second step, reverse transcription is carried out on the RNA template by adopting a Tiangen cDNA first strand synthesis kit
4. Polymerase chain reaction: the same experiment as the second step.
5. The statistical method comprises the following steps:
the results are expressed as mean ± sd and processed with SPSS16.0 statistical software. The average number comparison between the two groups adopts t test, and the difference is considered to have statistical significance when P is less than 0.05. The statistical plots were plotted using GraphPad prism5.0 software.
4. As a result:
the results are shown in fig. 2, compared with the control tissue, H19 expression was significantly up-regulated in intestinal tissue of 20 mice with severe scald, and the difference was statistically significant (P < 0.001), which is consistent with the chip detection results.
(III) LncRNA-H19 overexpression
The basic expression of H19 is very low in abundance, the expression is obviously increased after scalding, and lncRNA-H19 overexpression is carried out in rat small intestine crypt epithelial cells (IEC-6 cells).
1. Cell culture:
rat small intestinal crypt epithelial cells IEC-6 (purchased from Shanghai Meixuan Biotech Co., Ltd.) were cultured in RPMI1640 medium containing 10% fetal bovine serum and 1% P/S at 37 ℃ in 5% CO2Cultured in an incubator. The liquid was changed 1 time in 2 days. 0.25% trypsin digestion passage containing EDTA 10% DMSO + 90% fetal calf serum cryopreserved cells; cells in logarithmic growth phase were taken for experiments.
2. Construction of lncRNA-H19 overexpression adenovirus:
the over-expression adenovirus Ad-m-H19-EGFP is constructed by Hantaheng.
3. Infection:
IEC-6 cells were divided into 3 groups, each of which was a blank control group (IEC-6), a negative control group (Ad-m-EGFP control), and an experimental group (Ad-m-H19-EGFP), and the multiplicity of infection was MOI of 100.
4. Effect of infection with lncRNA-H19 on autophagy:
western Blot was used to test the effect on IEC-6 autophagy levels following infection with H19 overexpressing virus.
1) The cell culture was as above.
2) When the cells were in logarithmic growth phase, they were digested and passaged, on average, in three 10cm dis, at a density of 1 × 105/ml.
3) When the fusion degree of the three groups of cells is 50% -70%, adding control virus and over-expression virus according to MOI (100), wherein the volume of the culture medium is 1/2 of the normal culture volume, and supplementing the culture medium after infecting for 4-8 h.
4) Cells were harvested 48h after infection and three sets of cell proteins were extracted.
5) And (3) total protein extraction:
400ul of RIPA lysate was placed on ice and PMSF was added at a ratio of 100:1 within minutes before use to give a final concentration of 1mM PMSF.
② rinsing the cells for three times by precooled PBS, and sucking the PBS as dry as possible by a pipettor.
And thirdly, adding the prepared lysate into cells, scraping the cells by a cell scraper, transferring the cells into a 1.5ml centrifuge tube, fully vibrating the cells, fully cracking the cells on ice, vibrating the cells once every 5 minutes, and fully cracking the cells for 30 minutes.
Fourthly, centrifuging for 15min at the temperature of 4 ℃ and at the speed of 12000 g.
Transferring the supernatant total protein to a new 1.5ml centrifuge tube.
And sixthly, detecting the protein concentration by using the BCA method according to the detection instruction of the protein concentration in the Yunnan sky.
Seventhly, diluting the histone stock solutions to the same concentration by PBS, adding 5x protein loading buffer solution, boiling for 10 minutes to obtain protein loading solutions, wherein the loading amount of each group is 30ug, performing electrophoresis, electrotransformation, sealing, incubating primary antibody and secondary antibody according to the instructions, and developing the hypersensitive ECL chemiluminescent reagent.
5. Statistical analysis:
the experiments are completed according to 3 times of repetition, statistical analysis is carried out by SPSS16.0 statistical software, one-way analysis of variance (ANOVA) is adopted for comparison among groups, and P is less than 0.05, so that the statistical significance is realized.
6. As a result:
as shown in FIG. 3, compared with the blank control group and the negative control group, the autophagy level of the cells of the experimental group is obviously increased after the infection of Ad-m-H19-GFP, and the difference has statistical significance (P is less than 0.05), which indicates that H19 has the effect of improving the autophagy level of IEC-6 cells. The difference between the blank control group and the negative control group is not statistically significant.
The above description is only for the preferred embodiments of the present invention, but the protection scope of the present invention is not limited thereto, and any person skilled in the art can substitute or change the technical solution and the inventive concept of the present invention within the scope of the present invention.
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Claims (1)

1. Use of a reagent for detecting intestinal lncRNA H19 in preparation of a detection reagent for diagnosing severe burn.
CN201810210930.8A 2018-03-14 2018-03-14 Intestinal tissue marker lncRNAH19 after severe burn and application thereof Active CN108410874B (en)

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