CN111826433A - Application of LncRNA in diabetes prognosis evaluation and repeated abortion early warning - Google Patents
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Abstract
The invention discloses application of LncRNA in diabetes prognosis evaluation and repeated abortion early warning. The present invention provides an application of a substance for detecting LncRNA EPB41L4A-AS1 in (A1) or (A2) AS follows: (A1) preparing a product for diabetes prognosis evaluation or diabetes prognosis evaluation; (A2) preparing a product for early warning of repeated abortion or early warning of repeated abortion. Wherein, the nucleotide sequence of the LncRNA EPB41L4A-AS1 is shown AS SEQ ID No. 5. The invention indicates that the lncRNA EPB41l4A-AS1 has important significance for the prognosis evaluation of diabetes and the early warning of recurrent abortion.
Description
Technical Field
The invention relates to the technical field of biology, in particular to application of LncRNA in prognosis evaluation of diabetes and early warning of repeated abortion.
Background
The human genome contains a large number of DNA sequences that do not encode proteins, and RNA transcripts in which more than 80% of the non-coding sequences transcribe non-coding proteins are referred to as non-coding nucleic acids (ncrnas). Molecular weight greater than 200 nucleotides is called long noncoding nucleic acid (lncRNA). Recent studies have found that lncRNA plays an important role in regulating physiological and pathological processes of the body.
Diabetes is a common metabolic disease, and the incidence of diabetes is further increased along with the development of socioeconomic development in recent years. The international diabetes union promulgated in 2017 that worldwide diabetics reach 4.25 million, and the estimated possibility of 6.29 million by 2045, wherein Chinese diabetics can reach 1.144 million, and become the countries with the largest number of patients. Therefore, the prevention, diagnosis and treatment of diabetes have become important social problems to be solved urgently.
Recurrent abortion is more than 3 times of spontaneous abortion, and each abortion usually occurs in the same gestational month. The etiology of recurrent abortion is complex, and there are 43 diseases clinically which can finally cause recurrent abortion, including immune factors, hereditary factors, infectious factors, endocrine factors, anatomical factors and the like, wherein the abortion caused by immune factors accounts for 67% of the recurrent abortion.
Disclosure of Invention
The invention aims to provide application of LncRNA in diabetes prognosis evaluation and repeated abortion early warning.
In a first aspect, the present invention claims the use of a substance for detecting LncRNA EPB41L4A-AS1 in (a1) or (a2) AS follows:
(A1) preparing a product for diabetes prognosis evaluation or diabetes prognosis evaluation;
(A2) preparing a product for early warning of repeated abortion or early warning of repeated abortion.
In a second aspect, the invention claims the use of a substance for detecting LncRNA EPB41L4A-AS1 in any one of the following:
(a1) preparing a product for diagnosing the presence of the hyperglycemic stimulated aseptic inflammation or diagnosing the presence of the hyperglycemic stimulated aseptic inflammation;
(a2) preparing a product for early warning the aggravation of the diabetes or early warning the aggravation of the diabetes;
(a3) preparing a product for early warning of the vascular complications of diabetes, or early warning of the vascular complications of diabetes;
(a4) the product for early warning of the vascular endothelial oxidation injury of the diabetes is prepared, or the early warning of the vascular endothelial oxidation injury of the diabetes is carried out.
In diabetic patients, long-term vascular endothelial injury can cause vascular endothelial complications and further aggravate the diabetic condition.
In the aforementioned first and second aspects, the substance for detecting LncRNA EPB41L4A-AS1 may be a primer pair or a kit, or the like.
Wherein, the primer pair can be any one of the following primer pairs:
(b1) a primer pair consisting of two single-stranded DNAs shown as SEQ ID No.1 and SEQ ID No. 2;
(b2) and (b) a primer pair which consists of two single-stranded DNA molecules shown by sequences obtained by substituting and/or deleting and/or adding one or more nucleotides to SEQ ID No.1 and SEQ ID No.2, and has the same functions as the primer pair in (b 1).
In a specific embodiment of the invention, the kit is specifically a real-time fluorescent RT-PCR detection kit for detecting the LncRNA EPB41L4A-AS1, and contains the primer pair. Specifically, the kit contains a fluorescent RT-PCR reaction solution A and a fluorescent RT-PCR reaction solution B; the fluorescent RT-PCR reaction solution A contains DNA polymerase, dNTP, magnesium ions, a buffer system and the like; the fluorescent RT-PCR reaction solution B contains the primer pair, the fluorescent probe and the like.
In a third aspect, the invention claims the use of a substance for detecting LncRNA EPB41L4A-AS1 in any one of the following:
(c1) preparing a product for detecting the expression level of inflammatory factors in peripheral blood of a diabetic patient (e.g., type II diabetic patient), or detecting the expression level of inflammatory factors in peripheral blood of a diabetic patient (e.g., the expression level of inflammatory factors in mononuclear cells in peripheral blood);
(c2) preparing a product for measuring the expression level of the inflammatory factor in immune cells treated with high sugar (e.g., 30mM D-glucose) or LPS induction (e.g., 1. mu.g/ml LPS), or measuring the expression level of the inflammatory factor in immune cells treated with high sugar.
Further, the immune cell may be a monocyte or a lymphocyte.
In a particular embodiment of the invention, the monocytes are THP-1 cells; the lymphocyte is a Jurkat cell.
In (c1) and (c2), if the expression level of the LncRNA EPB41L4A-AS1 is measured to be lower than that of the normal control group, the inflammatory factor expression level is higher than or candidate to be higher than that of the normal control group. Wherein the normal control group can be healthy people without diabetes or immune cells without high sugar (or LPS induction) treatment.
In a fourth aspect, the invention claims the use of a substance for inhibiting LncRNA EPB41L4A-AS1 for the preparation of a cell model having the phenotype: intracellular inflammatory factor expression levels are elevated after treatment with high sugars (e.g., 30mM D-glucose) or LPS induction (e.g., 1. mu.g/ml LPS).
Further, the cell may be an immune cell; still further, the immune cell may be a monocyte; more specifically, the monocyte may be a THP-1 cell.
In a fifth aspect, the invention claims the use of a substance inhibiting LncRNA EPB41L4A-AS1 for the preparation of a product for enhancing oxidative damage in cells due to an inflammatory response, or for enhancing oxidative damage in cells due to an inflammatory response.
Wherein, the product can be a cell model or an animal model. Can be used for drug screening, etc.
Further, the cells may be villous cells (e.g., HTR-8/SVneo cells) or islet cells (e.g., Beta-TC-6 cells) or vascular endothelial cells (e.g., HUV-EC-C [ HUVEC ] cells).
In a sixth aspect, the present invention claims the use of immune cells with reduced expression levels of LncRNA EPB41L4A-AS1 for the preparation of a product for enhancing oxidative damage in cells due to inflammatory reactions, or for enhancing oxidative damage in cells due to inflammatory reactions.
Wherein, the product can be a cell model or an animal model. Can be used for drug screening, etc.
In a particular embodiment of the invention, the immune cell is in particular a THP-1 cell. Correspondingly, the immune cell with the reduced expression level of LncRNAEPB41L4A-AS1 is obtained by introducing siEPB41L4A-AS1 into THP-1 cells; the siEPB41L4A-AS1 is formed by annealing two single strands shown in SEQ ID No.3 and SEQ ID No. 4.
In the foregoing fourth and fifth aspects, the substance for inhibiting LncRNA EPB41L4A-AS1 may be specifically siEPB41L4A-AS 1; the siEPB41L4A-AS1 is formed by annealing two single-stranded RNAs shown in SEQ ID No.3 and SEQ ID No. 4.
In the foregoing fifth and sixth aspects, the enhancing of intracellular oxidative damage due to the inflammatory response may be embodied as increasing intracellular ROS levels due to the inflammatory response.
In a particular embodiment of the invention, the inflammatory response is in particular induced by high sugars (e.g.30 mM D-glucose) or LPS (the induction concentration of LPS may be 100-200 ng/ml).
In a seventh aspect, the invention also claims the primer pair or the kit as described above.
In the foregoing aspects, the LncRNA EPB41L4A-AS1 may be any one of the following:
(B1) RNA shown as SEQ ID No. 5;
(B2) RNA which is obtained by substituting and/or deleting and/or adding one or more nucleotide residues to the nucleotide sequence shown in SEQ ID No.5 and has the same function;
(B3) and (B) RNA having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology to the nucleotide sequence defined in (B1) or (B2) and having the same function.
In each of the foregoing aspects, the inflammatory factor may be as follows: IL1 beta, IL-8, TNF-alpha and/or IL-16.
The research of the invention finds that: IncRNA EPB41l4A-AS1 plays an important role in the development of diabetes. Compared with healthy control, the expression level of the peripheral blood nucleated cells LncRNA EPB41L4A-AS1 of the diabetic patients is obviously reduced. In contrast, the expression of inflammatory factors in the patient's peripheral blood nucleated cells was significantly increased. Prompting that there may be some connection between the two. Further research shows that LncRNA EPB41L4A-AS1 is an important regulatory molecule of peripheral blood nucleated cells, particularly monocyte and lymphocyte inflammatory factors, and high sugar stimulation can down-regulate the expression of LncRNA EPB41L4A-AS1 so AS to promote the expression of the monocyte and lymphocyte inflammatory factors. The lncRNA is also found to be abnormally expressed in the patients with the repeated abortion and possibly related to the abnormal immune state of the patients with the repeated abortion. The invention indicates that the lncRNA EPB41l4A-AS1 has important significance for the prognosis evaluation of diabetes and the early warning of recurrent abortion.
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FIG. 1 shows the expression levels of LncRNA EPB41L4A-AS1 in whole blood of patients with diabetes mellitus and recurrent abortion. A is diabetic; b is the patient with recurrent abortion and villus injury.
FIG. 2 is a bioinformatic analysis of the expression level of peripheral blood mononuclear inflammatory factors in diabetic patients with down-regulated expression of LncRNA EPB41L4A-AS 1. A is the expression of EPB41L4A-AS 1. B is the expression of IL-8. C is the expression of IL-6. D is the expression of IL-1 beta.
FIG. 3 shows that high-sugar and LPS treatment down-regulated the expression of LncRNA EPB41L4A-AS1 in monocytes and lymphocytes. A and B are respectively cultured in 30mM high sugar; c and D are 1. mu.g/mL LPS treatment, respectively.
FIG. 4 shows that the expression of inflammatory factors stimulated by high-sugar or LPS stimulation is significantly increased by comparison of the stably expressed down-regulated THP-1 cell line sh8 of LncRNA EPB41L4A-AS1 with a control cell line (shnc) expressing normal LncRNAEPB41L4A-AS 1. A is the expression of LncRNAEPB41L4A-AS1 of THP-1 cells (sh8) transfected with a plasmid stably expressing siEPB41L4A-AS1siRNA is remarkably reduced compared with a control group. B and C are marked by the increase of the expression of inflammatory factors stimulated under the stimulation of high sugar and LPS respectively.
FIG. 5 is a flow cytometer for detecting the effect of down-regulating LncRNA EPB41L4A-AS1 expression induced immune cell activation on oxidative damage of villus/islets/vascular endothelial cells.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
LncRNA EPB41L4A-AS1 referred to in the following examples is specifically LncRNA shown in SEQ ID No. 5.
Example 1 detection of expression of LncRNA EPB41L4A-AS1 in Whole blood
Sample supply group 1: healthy controls, clinically confirmed diabetic patients and patients with diabetes with tumor. The subjects informed and agreed.
Sample set 2: compared with the induced abortion group, the induced abortion group has repeated abortion with villus injury patients. The subjects informed and agreed.
Total RNA was extracted from fresh blood samples using the TRIZOL method. 1ml of RNAi assoplus (TAKARA) was added to 500. mu.l of whole blood, total RNA was obtained by chloroform extraction and isopropanol precipitation, concentration and mass analysis of total RNA was performed using a Nanodrop 2000 apparatus (Thermo Fisher Scientific), and total RNA was reverse transcribed into cDNA using a ReverTraace qPCR RT kit (TOYOBO).
The expression level of LncRNA EPB41L4A-AS1 was determined by fluorescent quantitative PCR using a quantitative gradient PCR apparatus qTOWER 2.2, Jena, Germany, using the SYBR GreenRealTime PCRMaster Mix (TOYOBO) system. Three replicates were made per sample using the β -actin gene as an internal reference.
The primers used for the detection of β -actin are as follows:
forward direction: 5'-TGACGTGGACATCCGCAAAG-3', respectively;
and (3) reversing: 5'-CTGGAAGGTGGACAGCGAGG-3' are provided.
The primers used for detecting LncRNA EPB41L4A-AS1 were AS follows:
forward direction: 5'-CCACCCTGAGTCTGGTGAGT-3' (SEQ ID No. 1);
and (3) reversing: 5'-CCTGGCATAGTCGATGATGTA-3' (SEQ ID No. 2).
As a result, AS shown in FIG. 1, the expression level of LncRNAEPB41L4A-AS1 in peripheral blood of diabetic patients and diabetic tumor-associated patients was significantly decreased AS compared with that of healthy controls (A in FIG. 1). Compared with the induced abortion group, the expression level of the repeated abortion patient is obviously reduced along with villus injury whole blood LncRNA EPB41L4A-AS1 (B in figure 1).
Example 2 bioinformatic analysis of expression levels of peripheral blood mononuclear inflammatory factor in diabetic patients with downregulation of expression of LncRNA EPB41L4A-AS1
Since the expression of the LncRNA EPB41L4A-AS1 in the peripheral blood is mainly from white blood cells, the expression of the LncRNA EPB41L4A-AS1 in the peripheral blood of the diabetic patients is analyzed by a bioinformatics method. The method comprises the following specific steps:
the expression level of LncRNA EPB41L4A-AS1 in peripheral blood mononuclear cells of normal persons (24 cases) and type II diabetics (12 cases) in the GDS3875 chips in GEO data sets is analyzed, and the expression level of LncRNA EPB41L4A-AS1 in peripheral blood mononuclear cells of type II diabetics is remarkably reduced. By analyzing the data in GDS3874 in the GEO database, the inflammatory factors IL-8, IL-6 and IL-1. beta. were found to be significantly increased in peripheral blood mononuclear cells of type II diabetic patients (12 cases) with reduced expression of LncRNA EPB41L4A-AS1 AS compared to healthy controls (24 cases) (FIG. 2).
Example 3 examination of the Effect of high sugar on the expression of LncRNA EPB41L4A-AS1 in monocytes and lymphocytes
(ii) THP-1 cells
TIB-202) and Jurkat cells
TIB-152) were separately cultured in serum-free 1640 medium containing 30 mMD-glucose for 0h, 2h, and 12h or in 1640 medium containing 1. mu.g/mL LPS and 10% Fetal Bovine Serum (FBS) (Biowest) for 0h, 2h, and 12h, total RNA in the cells was extracted by the method of example 1, and LncRNA was detected by qRT-PCRExpression of the EPB41L4A-AS1 gene.
As a result, it was found that: the expression level of LncRNA EPB41L4A-AS1 in monocyte-derived THP-1 cells and lymphocyte-derived JURKAT cells was significantly down-regulated at 12h of high-glucose and LPS treatment. As shown in fig. 3.
Example 4 establishment of THP-1 cell line with reduced expression of LncRNA EPB41L4A-AS1 and detection of inflammatory factor expression of the cell line under high sugar and LPS induction
Construction of a cell line stably overexpressing THP-1 of siRNA of specific LncRNAEPB41L4A-AS1
THP-1 human peripheral blood mononuclear cell leukemia cell line (ATCC company product of America
TIB-202) in 1640 medium containing 10% Fetal Bovine Serum (FBS) (Biowest corporation) under 5% carbon dioxide at 37 ℃.
DNA sequences encoding the siRNAs of negative control NC and siEPB41L4A-AS1 were annealed and ligated to lentiviral interference vector LV-2 (Shanghai Gilmama, C06002), respectively, to obtain overexpression plasmids stably expressing NC siRNA and siEPBL4A-AS1 siRNA.
Negative control NC siRNA sense strand: 5'-UUCUCCGAACGUGUCACGUTT-3', respectively;
negative control NC siRNA antisense strand: 5'-ACGUGACACGUUCGGAGAATT-3' are provided.
siEPB41L4A-AS1siRNA sense strand: 5'-GCCUGUCCUUUCUUCCUUUTT-3' (SEQ ID No. 3);
siEPB41L4A-AS1siRNA antisense strand: 5'-AAAGGAAGAAAGGACAGGCTT-3' (SEQ ID No. 4).
The plasmid is transfected into THP-1 cell line by Lipofectamine3000 (American Saimei Fei company, cargo number L3000001), the transfected cell line is changed into a fresh new culture medium after 6h, the culture medium is cultured for 48h, a complete culture medium containing 1 mug/ml puromycin 1640 is added for screening, and after about one month, the surviving cells are the THP-1 cells which stably express NC siRNA (shNC) or stably express siEPB41L4A-AS1siRNA (shepB41L4A-AS 1). Using the gene expression assay in a manner consistent with that of example 1, it was found that expression of LncRNAEPB41L4A-AS1 in THP-1 cells transfected with a stably expressing siEPB41L4A-AS1siRNA plasmid was significantly reduced compared to the control group, and the results are shown in A of FIG. 4.
Secondly, the expression of inflammatory factors under the induction of high sugar and LPS is detected by using the THP-1 cell strain with low expression of LncRNA EPB41L4A-AS1
The shNC and shEPB41L4A-AS1 cell lines of THP-1 were treated with 1640 medium containing 30mM D-glucose or 1. mu.g/mL LPS, respectively, for 12 hours, and then the expression levels of IL 1. beta., IL-8 and TNF in the cells were measured by the fluorescent quantitative PCR method in accordance with example 1.
Wherein, the primer pair for detecting the IL-1 beta gene expression is as follows:
forward direction: 5'-GCTACGAATCTCCGACCACCA-3', respectively;
and (3) reversing: 5'-GGGCAGGGAACCAGCATCT-3' are provided.
The primer pair for detecting the IL-8 gene expression is as follows:
forward direction: 5'-GACATACTCCAAACCTTTCCACCC-3', respectively;
and (3) reversing: 5'-AAAAACTTCTCCACAACCCTCTGC-3' are provided.
The primer pair for detecting TNF gene expression is
Forward direction: 5'-TGCTGCACTTTGGAGTGATC-3', respectively;
and (3) reversing: 5'-GGTTTGCTACAACATGGGCTA-3' are provided.
The results are shown in FIG. 4B and C, and the expression of IL1 beta, IL-8 and TNF-alpha, which are inflammatory factors stimulated by high sugar or LPS, is obviously increased.
Example 5 Effect of downregulation of immune cell activation induced by expression of LncRNA EPB41L4A-AS1 on oxidative damage of villus/islets/vascular endothelial cells
HTR-8(
CRL-3271TM)、beta-TC-6(
CRL-11506) and HUVEC (
CRL-1730) cells were all from ATCC, Inc., USA. HTR-8 and beta-TC-6 cells were cultured in 1640 medium (Thermo Fisher Scientific) containing 10% fetal bovine serum, and HUVEC cells were cultured in F-12K medium (Thermo Fisher Scientific) containing 10% fetal bovine serum at 37 ℃ under 5% carbon dioxide.
First, down-regulation of immune cell activation induced by expression of LncRNA EPB41L4A-AS1 on villus cells HTR-8/SVneo (
CRL-3271TM) Influence of oxidative damage of
1. The concentration of villous HTR8 cells in the logarithmic growth phase was adjusted to contain 20w cells per 2ml of cell suspension. 2ml of cell suspension was inoculated into 6well cell culture plates until cells were fully adherent.
2. The concentrations of the stable low expression LncRNA EPB41L4A-AS1 THP-1 cells (SHEPB41L4A-AS1) and the corresponding THP-1 cells (SHNC) AS a negative control in log phase were adjusted to contain about 100w cells in 2ml cell suspension. These two cells were constructed for example 4.
3. The culture solution of the villus cells HTR8 was removed, and 2ml of cells containing 100w of shNC or shEPB41L4A-AS1 THP-1 were added, respectively, to group the cells AS follows:
control group: mixing HTR8 cells and shNC cells, culturing, adding about 400ng LPS, and making the final concentration of LPS 200 ng/ml;
experimental groups: HTR8 cells were cultured in a mixed manner with SHEPB41L4A-AS1 cells, and about 400ng of LPS was added to give a final LPS concentration of 200 ng/ml.
The cells were incubated for 12h in a carbon dioxide incubator at 37 ℃.
4. The culture medium and the suspended THP-1 cells were removed, stained with 2ml of PBS containing 5. mu. mol of ROS probe for DCFH-DA (product of MCE, cat. No. HY-D0940) for 30min, the unstained DCFH-DA was washed off with PBS, the cells were collected, and the level of ROS in the package of HTR8 cells was detected by flow-assay.
The results are shown in FIG. 5. It can be seen that the THP-1 cells stably knocked down with LncRNA EPB41L4A-AS1 and HTR8 cells induced by 200ng/ml LPS for 12h were able to significantly increase the level of ROS in HTR8 cells, in response to oxidative damage of HTR8 cells.
Secondly, the immune cell activation induced by the expression of LncRNA EPB41L4A-AS1 is down-regulated to activate islet cells Beta-TC-6(
CRL-11506TM) Influence of oxidative damage of
1. The concentration of Beta-TC-6 in the islet cells in the logarithmic growth phase was adjusted to contain 15w cells per 2ml of cell suspension. 2ml of cell suspension was inoculated into 6well cell culture plates until cells were fully adherent.
2. The concentrations of the cells of THP-1 stably underexpressing LncRNA EPB41L4A-AS1 (SHEPB41L4A-AS1) and the corresponding negative control THP-1 (shNC) in log phase were adjusted to contain about 120w of cells in 2ml of cell suspension. These two cells were constructed for example 4.
3. The islet cell Beta-TC-6 culture solution was removed, and 2ml of THP-1 cells containing 120w of shNC or shepB41L4A-AS1 were added, respectively, and the cells were grouped AS follows:
control group: mixing islet cells Beta-TC-6 with shNC cells for culture, and adding about 400ng of LPS to make the final concentration of LPS be 200 ng/ml;
experimental groups: islet cells Beta-TC-6 were cultured in a mixed culture with shepB41L4A-AS1 cells, and about 400ng of LPS was added to give a final LPS concentration of 200 ng/ml.
The cells were incubated for 12h in a carbon dioxide incubator at 37 ℃.
4. The culture medium and the suspended THP-1 cells were removed, stained with 2ml of PBS containing 5. mu. mol of ROS probe for DCFH-DA (product of MCE, cat. No. HY-D0940) for 30min, the unstained DCFH-DA was washed off with PBS, the cells were collected, and the ROS level in Beta-TC-6 cells was detected by flow-assay.
The results are shown in FIG. 5. As can be seen, when THP-1 cells and islet cells Beta-TC-6 which are stably knocked down by LncRNA EPB41L4A-AS1 are induced for 12 hours by 200ng/ml LPS, the level of ROS in the islet cells Beta-TC-6 can be remarkably increased, and cells are responded to oxidative damage.
Thirdly, the immune cell activation induced by the expression of the LncRNA EPB41L4A-AS1 is reduced to activate the vascular endothelial cell HUV-EC-C [ HUVEC ]](
CRL-1730TM) The influence of oxidative damage of (c).
1. The HUVEC concentration of vascular endothelial cells in logarithmic growth phase was adjusted to contain 12w cells per 2ml cell suspension. 2ml of cell suspension was inoculated into 6well cell culture plates until cells were fully adherent.
2. The concentrations of the cells of THP-1 stably underexpressing LncRNA EPB41L4A-AS1 (SHEPB41L4A-AS1) and the corresponding negative control THP-1 (shNC) in log phase were adjusted to contain about 80w of cells in 2ml of cell suspension. These two cells were constructed for example 4.
3. The culture solution of vascular endothelial cells HUVEC was removed, and 2ml of cells containing 100w of shNC or shEPB41L4A-AS1 THP-1 were added, respectively, to group the cells AS follows:
control group: HUVEC and shNC cells were cultured in a mixed manner, and about 200ng of LPS was added to give a final LPS concentration of 100 ng/ml.
Experimental groups: HUVEC and SHEPB41L4A-AS1 cells were cultured in a mixed manner, and about 200ng of LPS was added to give a final LPS concentration of 100 ng/ml.
The cells were incubated for 12h in a carbon dioxide incubator at 37 ℃.
4. The culture medium and the suspended THP-1 cells were removed, stained with 2ml of PBS containing 5. mu. mol of ROS probe for DCFH-DA (product of MCE, cat. No. HY-D0940) for 30min, the unstained DCFH-DA was washed off with PBS, the cells were collected, and the ROS level in HUVEC cells of vascular endothelial cells was detected by flow-assay.
The results are shown in FIG. 5. As can be seen, when THP-1 cells and HUVEC of vascular endothelial cells, which are stably knocked down by LncRNA EPB41L4A-AS1, are induced by 100ng/ml LPS for 12 hours, the level of ROS in the HUVEC of the vascular endothelial cells can be remarkably increased, and severe oxidative damage of the HUVEC of the vascular endothelial cells is suggested.
Example 6 real-time fluorescent RT-PCR detection kit for detecting LncRNA EPB41L4A-AS1
This example provides a kit for the detection of LncRNA EPB41L4A-AS1 using real-time fluorescent RT-PCR.
[ MEASURING METHOD ]
Firstly, extracting nucleic acid
1. Take 200. mu.L of the treated sample, add 600. mu.L of lysate and 20. mu.L of proteinase K, mix them by turning upside down, and water bath at 55 ℃ for 10 min.
2. Absorbing all mixed liquid and transferring the mixed liquid into an adsorption column, centrifuging at 12,000rpm for 45s, discarding liquid in a collecting pipe, and sleeving the collecting pipe.
3. Add 600. mu.L of washing solution to the adsorption column, centrifuge at 12,000rpm for 45s, discard the liquid in the collection tube, and loop back the collection tube.
4. And (5) repeating the step (3).
5. The empty column was centrifuged at 12,000rpm for 2 min.
6. The adsorption column was transferred to a new nuclease-free 1.5mL centrifuge tube, 50. mu.L of the eluent was added to the center of the adsorption column membrane, and the column was allowed to stand at room temperature for 1min and centrifuged at 12,000rpm for 45s to obtain nucleic acid.
Two, real-time fluorescent RT-PCR
1. Preparing a reaction system: each fraction, 20. mu.L in total volume, was prepared as in Table 1 below, mixed well and labeled.
TABLE 1 reaction System
| Reagent | Dosage of |
| Fluorescent RT-PCR reaction solution A | 16.8μL |
| Fluorescent RT-PCR reaction solution B | 1.2μL |
| Form panel | 2.0μL |
Wherein the fluorescent RT-PCR reaction solution A comprises DNA polymerase, dNTP, magnesium ions, a buffer system and the like; the fluorescent RT-PCR reaction solution B comprises primer pairs, probes and the like shown in SEQ ID No.1 and SEQ ID No.2 in example 1.
And (4) flicking each tube, uniformly mixing, placing on an 8-tube centrifuge for instantaneous centrifugation for 10s, and throwing the liquid on the tube wall to the tube bottom.
2. And (3) fluorescent PCR reaction: the following reactions were performed on a fluorescent PCR instrument: 15min at 42 ℃ and 3min at 95 ℃; cycles of 95 ℃ for 5s and 58 ℃ for 40s, 40 times, and collects FAM fluorescent signals (reporter "FAM", quencher "None") in the second step of each cycle (58 ℃ for 40 s).
[ judgment of test results ]
When the sample to be detected has a typical amplification curve and the Ct value is less than or equal to 30.0, the sample is judged to be positive, and the sample without the Ct value is judged to be negative.
<110> Shenzhen research institute of Qinghua university, Shenzhen, Kangbaide Biotech limited;
<120> application of LncRNA in diabetes prognosis evaluation and repeated abortion early warning
<130>GNCLN190828
<160>5
<170>PatentIn version 3.5
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aaaggaagaa aggacaggct t 21
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acuggccaca cauccgcaga ggugacucgg gcugagaacc caggcuccuc cucccuuugg 180
ugacacgccc cucggucccu cacuggcacu ucucccuccg gccacacggc ggcgucucgc 240
cauagcgcag cggccgaugg uacagcccgc uccccccucg cgcucucgga cagugggucc 300
uuccacuugu agaaaagcau ugugggacgg aagccugucc uuucuuccuu uuggugcgag 360
cuugcugugg uuuuugcucu ggguccucug ggauggcgcc uggcuguggc cgcguggucu 420
cucacgcagg ggcgccgggc gggggaacgc ggccacccug agucugguga gucgacugcg 480
gcggccugug uccgaagugu ccggggccgu gaacaagggc agcggccugg ccucaggccu 540
gcguucccac guuuggaaac ggggagcuuc gucgauuugu guuuacauca ucgacuaugc 600
cagggaguuc uccagauaag ccugguuuua uuuucgucag ugaaaaggcc uuaccguaua 660
acugacuuua ugcuugcccu gcccccguau aaaauaacuu aaaagcagcg ugccugguua 720
cagcuguuuc cacgugcggu gcucgucggg agugaucacc uacccuacag gugaguuuuc 780
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ucggauuuua aaucgugaga aaauuucucu gaaauguauu guccguuuuu aggcuguaau 900
cggcauuacu gucagccagu cagcaaccuu augccauaaa gccuaccuca cgcaguguca 960
gccuuugugu ugcccauuca cuuuggaaac uagugaaugu ggugucaaaa aaggcguaaa 1020
uuaaacgcuu ugcagccuuu uccugcccuu aaauuugaua ccuuuggugu aggagcugca 1080
uaaguaacag uugcugcuuu uacguuucca cgcgugaucu ugacccugcu agccuuaagu 1140
guaugguuuc ucuuagccag uucuaauuuu uguucaggug gaagauggau gccugaagug 1200
uagacugcug cuagcugaau accaucuggg agcauaaagg ugaccugaag guagggugau 1260
augucuuaaa gcacuuugua augggaauuu uuaucaccuu uuaaauuggg guuccuucuc 1320
uagugaguuu uaaugucagu gguacauucg uaguguugcu cugucuguag cuauuaaggu 1380
gaguuaauaa augggauagc cuccacagcu uauuuuuggg aagguuuugc ugauacuucc 1440
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cuuaggaugu ccuuggugag gauuuugaaa auuugaucuu cacaagaguu gccuggauca 1680
uuugaaauuu cugggagucu gaggaguacu gacauaauua ccugcuggag ucuguaaaua 1740
cacauuuaag acagugagga ugugaauaaa uauauuaaug cacuuuggca uuuguguuuu 1800
aagugauuaa cugccagaaa cagcuauuuc uaaaaaguua uaagggagga gggguucuuu 1860
uuugaucagu auucacugcu gucaccauaa uuaauagccu uaaaauagcu uguguuuggc 1920
ccaaagagaa auguacuuuc uuccagugac ucaaaaaucg uggcuagaac uagacu 1976
Claims (10)
1. The use of a substance for detecting LncRNA EPB41L4A-AS1 in (A1) or (A2) AS follows:
(A1) preparing a product for diabetes prognosis evaluation or diabetes prognosis evaluation;
(A2) preparing a product for early warning of repeated abortion or early warning of repeated abortion.
2. The use of a substance for detecting LncRNA EPB41L4A-AS1 in any one of the following:
(a1) preparing a product for diagnosing the presence of the hyperglycemic stimulated aseptic inflammation or diagnosing the presence of the hyperglycemic stimulated aseptic inflammation;
(a2) preparing a product for early warning the aggravation of the diabetes or early warning the aggravation of the diabetes;
(a3) preparing a product for early warning of the vascular complications of diabetes, or early warning of the vascular complications of diabetes;
(a4) the product for early warning of the vascular endothelial oxidation injury of the diabetes is prepared, or the early warning of the vascular endothelial oxidation injury of the diabetes is carried out.
3. Use according to claim 1 or 2, characterized in that: the substance for detecting LncRNA EPB41L4A-AS1 is a primer pair or a kit;
the primer pair is any one of the following primer pairs:
(b1) a primer pair consisting of two single-stranded DNAs shown as SEQ ID No.1 and SEQ ID No. 2;
(b2) a primer pair which consists of two single-stranded DNA molecules shown by sequences obtained by substituting and/or deleting and/or adding one or more nucleotides to SEQ ID No.1 and SEQ ID No.2, and has the same function as the primer pair in (b 1);
the kit is a real-time fluorescent RT-PCR detection kit for detecting the LncRNA EPB41L4A-AS1, and contains the primer pair.
4. The use of a substance for detecting LncRNA EPB41L4A-AS1 in any one of the following:
(c1) preparing a product for detecting the expression level of the inflammatory factor in the peripheral blood of the diabetic patient, or detecting the expression level of the inflammatory factor in the peripheral blood of the diabetic patient;
(c2) preparing a product for detecting the expression level of the inflammatory factor in the immune cells subjected to high-sugar or LPS induction treatment, or detecting the expression level of the inflammatory factor in the immune cells subjected to high-sugar or LPS induction treatment;
further, the immune cell is a monocyte or lymphocyte;
still further, the monocytes are THP-1 cells; the lymphocyte is a Jurkat cell.
5. Use of a substance for inhibiting LncRNA EPB41L4A-AS1 for the preparation of a cell model having the phenotype: after high sugar or LPS induction treatment, the expression level of intracellular inflammatory factors is increased;
further, the cell is an immune cell;
further, the immune cell is a monocyte;
more specifically, the monocyte is a THP-1 cell.
6. Use of a substance for inhibiting LncRNA EPB41L4A-AS1 for the preparation of a product for enhancing oxidative damage in cells due to inflammatory response, or enhancing oxidative damage in cells due to inflammatory response;
further, the cells are villus cells or islet cells or vascular endothelial cells.
Use of immune cells with reduced LncRNA EPB41L4A-AS1 expression levels for the preparation of a product for enhancing oxidative damage in cells due to inflammatory response, or for enhancing oxidative damage in cells due to inflammatory response;
further, the immune cell is a THP-1 cell.
8. Use according to any one of claims 5 to 8, characterized in that: the substance for inhibiting LncRNA EPB41L4A-AS1 of claims 5 and 6 is siEPB41L4A-AS 1; the siEPB41L4A-AS1 is formed by annealing two single-stranded RNAs shown in SEQ ID No.3 and SEQ ID No. 4; and/or
In claims 6 and 7, said enhancement of intracellular oxidative damage due to inflammatory response is manifested by an increase of intracellular ROS levels due to inflammatory response.
9. The primer pair or the kit according to claim 3.
10. The use or kit according to any one of claims 1 to 9, wherein: the LncRNAEPB41L4A-AS1 is any one of the following components:
(B1) RNA shown as SEQ ID No. 5;
(B2) RNA which is obtained by substituting and/or deleting and/or adding one or more nucleotide residues to the nucleotide sequence shown in SEQ ID No.5 and has the same function;
(B3) RNA having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology to the nucleotide sequence defined in (B1) or (B2) and having the same function;
and/or
The inflammatory factors are as follows: IL1 beta, IL-8, TNF-alpha and/or IL-16.
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| CN112575076A (en) * | 2020-11-02 | 2021-03-30 | 深圳恒生医院 | LncRNA marker for detecting and evaluating early spontaneous abortion and product using LncRNA marker |
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