CN111826433B - Application of LncRNA in prognosis evaluation of diabetes and early warning of recurrent abortion - Google Patents
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Abstract
The invention discloses application of LncRNA in prognosis evaluation of diabetes and early warning of recurrent abortion. The invention provides the use of a substance for detecting LncRNA EPB41L4A-AS1 in (A1) or (A2) aS follows: (A1) Preparing a product for prognosis evaluation of diabetes, or prognosis evaluation of diabetes; (A2) Preparing a product for repeated abortion early warning or repeated abortion early warning. Wherein the nucleotide sequence of LncRNA EPB41L4A-AS1 is shown aS SEQ ID No. 5. The invention prompts that the lncRNA EPB41l4A-AS1 has important significance for prognosis evaluation of diabetes and 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 recurrent abortion.
Background
There are a large number of DNA sequences in the human genome that do not encode proteins, of which more than 80% of the non-coding sequences transcribe RNA transcripts of non-coding proteins, called non-coding nucleic acids (ncRNA). A long non-coding nucleic acid (lncRNA) is referred to as having a molecular weight greater than 200 nucleotides. Recent studies have found that lncRNA plays an important role in regulating physiological and pathological processes in the body.
Diabetes is a common metabolic disease, and the incidence of diabetes has further increased in recent years with the development of socioeconomic performance. Global diabetics published by the international diabetes consortium in 2017 have reached 4.25 billion and could be expected to reach 6.29 billion by 2045. Therefore, the prevention, diagnosis and treatment of diabetes become important social problems to be solved urgently.
Recurrent abortion is a natural abortion with more than 3 consecutive times, and each abortion usually occurs in the same gestational month. The cause of recurrent abortion is complex, 43 diseases can finally cause recurrent abortion clinically, and the recurrent abortion comprises immunity factors, hereditary factors, infectious factors, endocrine factors, anatomic factors and the like, wherein the abortion caused by the immunity factors accounts for 67% of recurrent abortion.
Disclosure of Invention
The invention aims to provide an application of LncRNA in prognosis evaluation of diabetes and early warning of recurrent abortion.
In a first aspect, the invention claims the use of a substance for detecting LncRNA EPB41L4A-AS1 in (A1) or (A2) aS follows:
(A1) Preparing a product for prognosis evaluation of diabetes, or prognosis evaluation of diabetes;
(A2) Preparing a product for repeated abortion early warning or repeated abortion early warning.
In a second aspect, the invention claims the use of a substance for detecting LncRNA EPB41L4A-AS1 in any of the following:
(a1) Preparing a product for diagnosing the presence or absence of aseptic inflammation of hyperglycemic stimulus, or diagnosing the presence or absence of aseptic inflammation of hyperglycemic stimulus;
(a2) Preparing a product for pre-warning the aggravation of diabetes, or pre-warning the aggravation of diabetes;
(a3) Preparing a product for early warning the vascular complications of diabetes or early warning the vascular complications of diabetes;
(a4) The method is used for preparing products for early warning the vascular endothelial oxidative damage of diabetes or early warning the vascular endothelial oxidative damage of diabetes.
For diabetics, long-term vascular endothelial injury can cause vascular endothelial complications and further exacerbate the diabetic condition.
In the foregoing 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 in SEQ ID No.1 and SEQ ID No. 2;
(b2) Consists of two single-stranded DNA molecules shown in sequences obtained by substituting and/or deleting and/or adding one or more nucleotides of SEQ ID No.1 and SEQ ID No.2, and has the same function 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 comprises the primer pair. Specifically, the kit contains fluorescent RT-PCR reaction liquid A and fluorescent RT-PCR reaction liquid 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 liquid B contains the primer pair, a fluorescent probe and the like.
In a third aspect, the invention claims the use of a substance for detecting LncRNA EPB41L4A-AS1 in any of the following:
(c1) Preparing a product for detecting the expression level of inflammatory factors in peripheral blood of a diabetic patient (such as a type II diabetic patient), or detecting the expression level of inflammatory factors in peripheral blood of a diabetic patient (such as the expression level of inflammatory factors in peripheral blood mononuclear cells);
(c2) Products were prepared for detecting the level of inflammatory factor expression in immune cells treated with high sugar (e.g., 30mM D-glucose) or LPS induction (e.g., 1. Mu.g/ml LPS), or in immune cells treated with high sugar.
Further, the immune cells may be monocytes or lymphocytes.
In a specific embodiment of the invention, the monocytes are THP-1 cells; the lymphocyte is a Jurkat cell.
In (c 1) and (c 2), if the expression level of the LncRNA EPB41L4A-AS1 is measured to be lower than that of the normal control group, the expression level of the inflammatory factor is higher or is candidate to be higher than that of the normal control group. Wherein the normal control group may be, for example, healthy people not suffering from diabetes or immune cells not treated with high sugar (or LPS induction).
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 following phenotype: intracellular inflammatory factor expression levels are elevated following treatment with high sugar (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 cells may be monocytes; more specifically, the monocytes may be THP-1 cells.
In a fifth aspect, the invention claims the use of a substance for inhibiting LncRNA EPB41L4A-AS1 for the preparation of a product for enhancing intracellular oxidative damage due to an inflammatory response, or for enhancing intracellular oxidative damage due to an inflammatory response.
Wherein the product may be a cell model or an animal model. Can be used for drug screening and the like.
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 invention claims the use of immune cells having reduced expression levels of LncRNA EPB41L4A-AS1 for the preparation of a product for enhancing intracellular oxidative damage due to an inflammatory response, or for enhancing intracellular oxidative damage due to an inflammatory response.
Wherein the product may be a cell model or an animal model. Can be used for drug screening and the like.
In a specific embodiment of the invention, the immune cells are in particular THP-1 cells. Correspondingly, the immune cells with reduced expression level of LncRNA EPB41L4A-AS1 are specifically 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 specifically be siEPB41L4A-AS1; the siEPB41L4A-AS1 is formed by annealing two single-stranded RNAs shown in SEQ ID No.3 and SEQ ID No. 4.
In the fifth and sixth aspects of the foregoing, the enhancing intracellular oxidative damage due to an inflammatory response may be embodied as increasing intracellular ROS levels due to an inflammatory response.
In a specific embodiment of the invention, the inflammatory response is specifically induced by high sugar (e.g. 30mM D-glucose) or LPS (the induction concentration of LPS may be 100-200 ng/ml).
In a seventh aspect, the invention also claims a primer pair as described hereinbefore or a kit as described hereinbefore.
In the foregoing aspects, the LncRNA EPB41L4A-AS1 may be any of the following:
(B1) RNA shown in SEQ ID No. 5;
(B2) RNA with the same function obtained by substituting and/or deleting and/or adding one or more nucleotide residues of the nucleotide sequence shown in SEQ ID No. 5;
(B3) RNA having a homology of 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more with the nucleotide sequence defined in (B1) or (B2) and having the same function.
In 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: lncRNA EPB41l4A-AS1 plays an important role in the development and progression of diabetes. The expression level of LncRNA EPB41L4A-AS1 in peripheral blood of diabetic patients was significantly decreased compared to healthy controls. In response, the expression of inflammatory factors in peripheral blood nucleated cells of the patient is significantly increased. Suggesting that there may be some relationship between the two. Further research has found that LncRNA EPB41L4A-AS1 is an important regulatory molecule of peripheral blood nucleated cells, especially mononuclear cells and lymphocyte inflammatory factors, and that high sugar stimulation can enable the expression of LncRNA EPB41L4A-AS1 to be down-regulated, thereby promoting the expression of the mononuclear cells and lymphocyte inflammatory factors. The study also found that the lncRNA was abnormally expressed in patients with recurrent abortion, possibly in relation to abnormal immune status in patients with recurrent abortion. The invention prompts that the lncRNA EPB41l4A-AS1 has important significance for prognosis evaluation of diabetes and early warning of recurrent abortion.
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FIG. 1 shows the expression level of LncRNA EPB41L4A-AS1 in whole blood of patients with diabetes and recurrent abortion. A is diabetes patient; b is a patient with recurrent abortion and villus injury.
FIG. 2 is a bioinformatic analysis of peripheral blood mononuclear cell inflammatory factor expression levels in diabetics 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. And the like.
FIG. 3 is a graph showing that high sugar and LPS treatment down-regulates expression of LncRNA EPB41L4A-AS1 in monocytes and lymphocytes. A and B are 30mM high sugar culture respectively; c and D were 1. Mu.g/mL LPS treatment, respectively.
FIG. 4 shows that the down-regulated THP-1 cell line sh8 with stable expression of LncRNA EPB41L4A-AS1 significantly increased expression of inflammatory factors stimulated by high sugar or LPS compared to a control cell line (shnc) with normal expression of LncRNA EPB41L4A-AS 1. A is that the expression of LncRNA EPB41L4A-AS1 of THP-1 cells (sh 8) transfected with the siRNA plasmid stably expressing siEPB41L4A-AS1 is significantly reduced compared with the control group. B and C are significantly increased in the expression of stimulatory inflammatory factors upon stimulation with high sugar and LPS, respectively.
FIG. 5 is a graph showing the effect of flow cytometry detection on oxidative damage to villus/islets/vascular endothelial cells of immune cell activation induced by downregulation of LncRNA EPB41L4A-AS1 expression.
Detailed Description
The experimental methods used in the following examples are conventional methods unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The LncRNA EPB41L4A-AS1 referred to in the following examples is specifically LncRNA shown in SEQ ID No. 5.
Example 1 detection of Whole blood LncRNA EPB41L4A-AS1 expression
Sample supply group 1: healthy controls, clinically established diabetics and diabetics with tumors. The test subjects informed and agreed.
Sample supply group 2: comparing the artificial abortion group, repeating abortion with villus injury patients. The test subjects informed and agreed.
Total RNA was extracted from fresh blood samples using the TRIZOL method. 1ml of RNAiso plus (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 the total RNA was performed by a Nanodrop 2000 instrument (Thermo Fisher Scientific), and the total RNA was reverse transcribed into cDNA by ReverTraAce qPCR RT kit (TOYOBO).
The expression level of LncRNA EPB41L4A-AS1 was detected by a fluorescent quantitative PCR method, and was detected by a fluorescent quantitative gradient PCR instrument qTOWER 2.2 in Yes, germany using a SYBR Green RealTime PCRMaster Mix (TOYOBO) system. The betA-Actin gene was used as an internal control, and three replicates were made per sample.
The primers used to detect betA-Actin were as follows:
forward direction: 5'-TGACGTGGACATCCGCAAAG-3';
reversing: 5'-CTGGAAGGTGGACAGCGAGG-3'.
The primers used for detection of LncRNA EPB41L4A-AS1 were aS follows:
forward direction: 5'-CCACCCTGAGTCTGGTGAGT-3' (SEQ ID No. 1);
reversing: 5'-CCTGGCATAGTCGATGATGTA-3' (SEQ ID No. 2).
As a result, aS shown in FIG. 1, the expression level of LncRNA EPB41L4A-AS1 was significantly decreased in peripheral blood of diabetic patients and diabetic tumor-associated patients aS compared with healthy controls (A in FIG. 1). Compared with the artificial abortion group, the expression level of LncRNA EPB41L4A-AS1 in the whole blood with villus damage of the patient with recurrent abortion was significantly decreased (B in FIG. 1).
Example 2 bioinformatic analysis of expression levels of peripheral blood mononuclear cell inflammatory factors in diabetics with downregulated expression of LncRNA EPB41L4A-AS1
Since the expression of LncRNAEPB41L4A-AS1 in peripheral blood is mainly derived from leucocytes, the expression of LncRNAEPB41L4A-AS1 in peripheral blood of diabetic patients is analyzed by the bioinformatics method. The method comprises the following steps:
through the analysis of the expression level of LncRNAEPB41L4A-AS1 in peripheral blood mononuclear cells of normal people (24 cases) and type II diabetes patients (12 cases) in GDS3875 chips in GEO dataset, the expression level of LncRNAEPB41L4A-AS1 in peripheral blood mononuclear cells of type II diabetes patients is found to be significantly reduced. By analysis of the data in GDS3874 in the GEO database, it was found that the inflammatory factors IL-8, IL-6 and IL-1β were significantly elevated in peripheral blood mononuclear cells of type II diabetics (12 cases) with reduced expression of LncRNA EPB41L4A-AS1 compared to healthy controls (24 cases) (FIG. 2).
Example 3 detection of Effect of high sugar on monocyte and lymphocyte LncRNA EPB41L4A-AS1 expression
THP-1 cells (ATCC tiB-202) and Jurkat cells (ATCC tiB-152) were stimulated with serum-free 1640 medium containing 30mM D-glucose for 0h,2h,12h, or with 1640 medium containing 1. Mu.g/mL LPS and 10% Fetal Bovine Serum (FBS) (Biowest Co.) for 0h,2h,12h, respectively, total RNA was extracted from the cells by the method of example 1, and qRT-PCR was performed to detect expression of LncRNAEPB41L4A-AS1 gene.
The result shows that: at 12h of high sugar and LPS treatment, the expression level of LncRNAEPB41L4A-AS1 in both monocyte-derived THP-1 cells and lymphocyte-derived Jurkat cells had been significantly down-regulated. As shown in fig. 3.
EXAMPLE 4 establishment of THP-1 cell line knocked down LncRNA EPB41L4A-AS1 expression, detection of the expression of inflammatory factors under high sugar and LPS Induction by the cell line
1. Construction of a cell line of THP-1 that stabilizes siRNA over-expressing specific LncRNAEPB41L4A-AS1
The THP-1 human peripheral blood mononuclear cell leukemia cell line was ATCC (ATCC tiB-202) and the medium was 1640 medium (Thermo Fisher Scientific) containing 10% Fetal Bovine Serum (FBS) (Biowest Co.) and the culture conditions were 37℃and 5% carbon dioxide.
DNA sequences encoding siRNA of negative control NC and siEPB41L4A-AS1 were annealed and ligated into lentiviral interference vector LV-2 (Shanghai Ji Ma Co., C06002) respectively to obtain overexpression plasmids stably expressing NC siRNA and siEPBL4A-AS1 siRNA.
Negative control NC siRNA sense strand: 5'-UUCUCCGAACGUGUCACGUTT-3';
negative control NC siRNA antisense strand: 5'-ACGUGACACGUUCGGAGAATT-3'.
siEPB41L4A-AS1 siRNA sense strand: 5'-GCCUGUCCUUUCUUCCUUUTT-3' (SEQ ID No. 3);
siEPB41L4A-AS1 siRNA antisense strand: 5'-AAAGGAAGAAAGGACAGGCTT-3' (SEQ ID No. 4).
The plasmid was transfected into THP-1 cell line with Lipofectamine3000 (Sieimer, inc., cat# L3000001), after 6 hours of transfection, fresh new medium was changed, after 48 hours of culture, complete medium containing 1. Mu.g/ml puromycin 1640 was added for selection, and after about one month, surviving cells were THP-1 cells stably expressing NC siRNA (shNC) or siEPB41L4A-AS1 siRNA (shEPB 41L4A-AS 1). Gene expression was examined in the same manner aS in example 1 and it was found that the expression of LncRNA EPB41L4A-AS1 was significantly reduced in THP-1 cells transfected with the siRNA plasmid stably expressing siEPB41L4A-AS1, compared with the control group, and the results are shown in FIG. 4A.
2. Using the above THP-1 cell line with low expression of LncRNA EPB41L4A-AS1, the expression of inflammatory factor under high sugar and LPS induction was examined
After the THP-1 shNC and shEPB41L4A-AS1 cell lines were treated with 1640 medium containing 30mM D-glucose or 1. Mu.g/mL LPS, respectively, for 12 hours, the intracellular IL 1. Beta., IL-8 and TNF expression levels were detected by the fluorescent quantitative PCR method in accordance with that in example 1.
Wherein, the primer pair for detecting IL-1 beta gene expression is:
forward direction: 5'-GCTACGAATCTCCGACCACCA-3';
reversing: 5'-GGGCAGGGAACCAGCATCT-3'.
Primer pairs for detecting IL-8 gene expression are:
forward direction: 5'-GACATACTCCAAACCTTTCCACCC-3';
reversing: 5'-AAAAACTTCTCCACAACCCTCTGC-3'.
Primer pairs for detecting TNF gene expression are
Forward direction: 5'-TGCTGCACTTTGGAGTGATC-3';
reversing: 5'-GGTTTGCTACAACATGGGCTA-3'.
The results are shown in FIGS. 4B and C, where the expression of the inflammatory factors IL 1. Beta., IL-8 and TNF-alpha were significantly increased upon stimulation with high sugar or LPS.
Example 5 Effect of immune cell activation induced by Down-regulating LncRNA EPB41L4A-AS1 expression on oxidative damage to villus/islets/vascular endothelial cells
HTR-8 (ATCC cube CRL-3271 ™), beta-TC-6 (ATCC cube CRL-11506) and HUVEC (ATCC cube CRL-1730) cells were all from ATCC company of America. 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.
1. Downregulation of Effect of immune cell activation induced by LncRNA EPB41L4A-AS1 expression on oxidative damage of villous cells HTR-8/SVneo (ATCC CRL-3271 ™)
1. The concentration of the villus cells HTR8 cells in the logarithmic growth phase was adjusted to contain 20w cells per 2ml of cell suspension. 2ml of the cell suspension was inoculated into a 6 well cell culture plate until the cells were completely adherent.
2. The concentrations of THP-1 cells (shEPB 41L4A-AS 1) stably and lowly expressing LncRNA EPB41L4A-AS1 in the logarithmic phase and corresponding THP-1 cells (shNC) aS a negative control were adjusted so that about 100w of cells were contained in 2ml of the cell suspension. These two cells were constructed for example 4.
3. Culture medium from which villus cells HTR8 were removed was added with 2ml of cells containing 100w of THP-1 of shNC or shEPB41L4A-AS1, respectively, and the cells were grouped aS follows:
control group: HTR8 cells and shNC cells are mixed and cultured, and about 400ng of LPS is added, so that the final concentration of the LPS is 200ng/ml;
experimental group: HTR8 cells were mixed with shEPB41L4A-AS1 cells and cultured, and about 400ng of LPS was added thereto so that the final concentration of LPS was 200ng/ml.
The cells were incubated in a carbon dioxide incubator at 37℃for 12h.
4. The culture broth and suspended THP-1 cells were removed, stained with 2ml of PBS solution containing 5. Mu. Mol of DCFH-DA ROS probe (product of MCE company, product number HY-D0940), washed with PBS to remove the uninfected DCFH-DA dye, the cells were collected, and the level of ROS in the bag of HTR8 cells was detected by flow.
The results are shown in FIG. 5. It can be seen that after THP-1 cells and HTR8 cells stably knocked down with LncRNA EPB41L4A-AS1 are induced for 12 hours in LPS of 200ng/ml, the level of ROS in the HTR8 cells can be remarkably increased, and the HTR8 cells are subjected to oxidative damage.
2. Down-regulating influence of immune cell activation induced by LncRNA EPB41L4A-AS1 expression on oxidative damage of islet cells Beta-TC-6 (ATCC [ Beta ] [ CRL ] -11506 ™)
1. The concentration of islet cells Beta-TC-6 in the logarithmic growth phase was adjusted to contain 15w cells per 2ml of cell suspension. 2ml of the cell suspension was inoculated into a 6 well cell culture plate until the cells were completely adherent.
2. The concentration of THP-1 cells (shEPB 41L4A-AS 1) stably and lowly expressing LncRNA EPB41L4A-AS1 in the logarithmic phase and the corresponding negative control THP-1 cells (shNC) were adjusted so that about 120w of cells were contained in 2ml of the cell suspension. These two cells were constructed for example 4.
3. Culture medium of islet cells Beta-TC-6 was removed, 2ml of cells containing 120w of THP-1 of shNC or shEPB41L4A-AS1 were added, and the cells were grouped aS follows:
control group: islet cells Beta-TC-6 and shNC cells are mixed and cultured, and about 400ng of LPS is added, so that the final concentration of the LPS is 200ng/ml;
experimental group: islet cells Beta-TC-6 were mixed with shEPB41L4A-AS1 cells, and approximately 400ng of LPS was added to give a final LPS concentration of 200ng/ml.
The cells were incubated in a carbon dioxide incubator at 37℃for 12h.
4. The culture broth and suspended THP-1 cells were removed, stained with 2ml of PBS solution containing 5. Mu. Mol of DCFH-DA as ROS probe (product of MCE company, product number HY-D0940), washed with PBS to remove the uninfected DCFH-DA dye, the cells were collected, and the level of ROS in Beta-TC-6 cells was detected by flow.
The results are shown in FIG. 5. It can be seen that when THP-1 cells of LncRNA EPB41L4A-AS1 and islet cells Beta-TC-6 are stably knocked down and induced for 12 hours in LPS of 200ng/ml, the level of ROS in the islet cells Beta-TC-6 can be remarkably increased, and reactive cells have oxidative damage.
3. Down-regulating the effect of immune cell activation induced by LncRNA EPB41L4A-AS1 expression on oxidative damage of vascular endothelial cells HUV-EC-C [ HUVEC ] (ATCC cube CRL-1730 ™).
1. The concentration of vascular endothelial cells HUVEC in the logarithmic growth phase was adjusted to contain 12w cells per 2ml of cell suspension. 2ml of the cell suspension was inoculated into a 6 well cell culture plate until the cells were completely adherent.
2. The concentration of THP-1 cells (shEPB 41L4A-AS 1) stably and lowly expressing LncRNA EPB41L4A-AS1 in the logarithmic phase and the corresponding negative control THP-1 cells (shNC) were adjusted so that about 80w of cells were contained in 2ml of the cell suspension. These two cells were constructed for example 4.
3. Culture medium of vascular endothelial cells HUVEC was removed, and 2ml of cells containing 100w of THP-1 of shNC or shEPB41L4A-AS1 were added, respectively, and the cells were grouped aS follows:
control group: vascular endothelial cells HUVEC were cultured in admixture with shNC cells, and about 200ng of LPS was added to give a final concentration of 100ng/ml.
Experimental group: vascular endothelial cells HUVEC were cultured in admixture with shEPB41L4A-AS1 cells, and about 200ng of LPS was added to give a final LPS concentration of 100ng/ml.
The cells were incubated in a carbon dioxide incubator at 37℃for 12h.
4. The culture broth and suspended THP-1 cells were removed, stained with 2ml of PBS solution containing 5. Mu. Mol of DCFH-DA as ROS probe (product of MCE company, product number HY-D0940), washed with PBS to remove the uninfected DCFH-DA dye, the cells were collected, and the level of ROS in the vascular endothelial cells HUVEC cells was detected by flow.
The results are shown in FIG. 5. It can be seen that when THP-1 cells of LncRNA EPB41L4A-AS1 and vascular endothelial cells HUVEC are stably knocked down and induced for 12 hours by LPS of 100ng/ml, the level of ROS in the vascular endothelial cells HUVEC can be remarkably increased, which suggests that the oxidative damage of the vascular endothelial cells HUVEC is severe.
Example 6 real-time fluorescent RT-PCR detection kit for detecting LncRNA EPB41L4A-AS1
The embodiment provides a kit for detecting LncRNA EPB41L4A-AS1 by using real-time fluorescence RT-PCR.
[ detection method ]
1. Nucleic acid extraction
1. 200. Mu.L of the treated sample was taken, 600. Mu.L of the lysate and 20. Mu.L of proteinase K were added, mixed upside down and in a water bath at 55℃for 10min.
2. All the mixed liquid is sucked and transferred into an adsorption column, centrifuged for 45s at 12,000rpm, the liquid in the collecting pipe is discarded, and the collecting pipe is sleeved.
3. 600. Mu.L of the washing solution was added to the column, centrifuged at 12,000rpm for 45s, the liquid in the collection tube was discarded, and the collection tube was returned.
4. And (3) repeating the step 3.
5. The column was centrifuged at 12,000rpm for 2min.
6. The adsorption column was transferred to a new 1.5mL centrifuge tube without nuclease, 50. Mu.L of the eluent was added to the center of the membrane of the adsorption column, and the mixture was allowed to stand at room temperature for 1min and centrifuged at 12,000rpm for 45s to obtain nucleic acid.
2. Real-time fluorescent RT-PCR
1. Preparing a reaction system: each 20 mu L total volume was prepared as in Table 1 below, thoroughly mixed and marked.
TABLE 1 reaction system
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 liquid B comprises primer pairs, probes and the like shown in SEQ ID No.1 and SEQ ID No.2 in the embodiment 1.
And (3) mixing each tube slightly, putting the mixture on an 8-tube-connected centrifuge, and carrying out instantaneous centrifugation for 10s to throw the liquid on the tube wall to the bottom of the tube.
2. Fluorescent PCR reaction: the following reactions were performed on a fluorescent PCR instrument: 15min at 42 ℃ and 3min at 95 ℃; FAM fluorescence signals (reporter group "FAM", quencher group "None") were collected in the second step of each cycle (58 ℃ C. 40 s) by cycling at 95 ℃ C. For 5s, 58 ℃ C. For 40s, 40 total times.
[ judgment of test results ]
When a typical amplification curve appears in the sample to be detected and the Ct value is less than or equal to 30.0, the sample to be detected is judged to be positive, and no Ct value is judged to be negative.
<110> Shenzhen research institute, shenzhen City Kangbai Biotechnology Co., ltd;
<120> application of LncRNA in prognosis evaluation of diabetes and early warning of recurrent abortion
<130> GNCLN190828
<160> 5
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213> Artificial sequence
<400> 1
ccaccctgag tctggtgagt 20
<210> 2
<211> 21
<212> DNA
<213> Artificial sequence
<400> 2
cctggcatag tcgatgatgt a 21
<210> 3
<211> 21
<212> RNA
<213> Artificial sequence
<400> 3
gccuguccuu ucuuccuuut t 21
<210> 4
<211> 21
<212> RNA
<213> Artificial sequence
<400> 4
aaaggaagaa aggacaggct t 21
<210> 5
<211> 1976
<212> RNA
<213> Artificial sequence
<400> 5
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ucauaugcuc uugacccugg ucgggugggg ggaugucugg auaccuccuc uguggaaaga 120
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
acguucgugc aagaccaguc gccauuuaaa acgcaucgca uuucacuuuu cauuauuaag 840
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
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uagugaguuu uaaugucagu gguacauucg uaguguugcu cugucuguag cuauuaaggu 1380
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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 (3)
1. The application of the substances for quantitatively detecting LncRNA EPB41L4A-AS1 in whole blood in preparing products for repeated abortion with villus injury early warning;
the substance is a primer pair or a kit;
the primer pair consists of two single-stranded DNA shown in SEQ ID No.1 and SEQ ID No. 2;
the kit is a real-time fluorescent RT-PCR detection kit for detecting the LncRNA EPB41L4A-AS1, and contains the primer pair.
2. Use of a primer pair for quantitatively detecting LncRNA EPB41L4A-AS1 in whole blood in the preparation of a product for detecting the expression level of inflammatory factors in peripheral blood of a diabetic patient;
if the expression level of LncRNA EPB41L4A-AS1 is lower than that of the normal control group, the expression level of the inflammatory factor is higher than or the candidate is higher than that of the normal control group; the normal control group is healthy people without diabetes; the inflammatory factors are as follows: IL-6, IL-8 and/or IL-1β;
the primer pair consists of two single-stranded DNA shown in SEQ ID No.1 and SEQ ID No. 2.
3. Use of a substance for inhibiting LncRNA EPB41L4A-AS1 in THP-1 of monocyte origin for the preparation of a cell model having the following phenotype: after induction treatment by high sugar or LPS, the intracellular inflammatory factor expression level is increased;
the inflammatory factor is IL-1 beta, IL-8 and/or TNF-alpha; the substance for inhibiting LncRNA EPB41L4A-AS1 is siEPB41L4A-AS1; the siEPB41L4A-AS1 is formed by annealing two single-stranded RNAs shown in SEQ ID No.3 and SEQ ID No. 4.
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