CN114438187A - MiRNA marker for diagnosing early abortion and application thereof - Google Patents
MiRNA marker for diagnosing early abortion and application thereof Download PDFInfo
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Abstract
The invention discloses a novel biomarker for early abortion diagnosis, and particularly relates to a method and application of hsa-miR-29a-3p in diagnosis, prediction and/or monitoring of early abortion. The invention firstly provides that hsa-miR-29a-3p is used as a marker for early pregnancy embryo implantation and poor pregnancy outcome, and then an in-vitro endometrial stromal cell decidualization model is used for verifying the decidualization regulation and control effect of hsa-miR-29a-3 p. The spatial-temporal expression specificity based on the marker has wide clinical diagnosis and prediction application prospects. The specific expression of the hsa-miR-29a-3p in different gestation periods and decidua tissues of early abortion can effectively predict and diagnose early abortion of pregnancy.
Description
Technical Field
The invention belongs to the field of biological detection and diagnosis, and particularly relates to a miRNA biomarker for early abortion diagnosis of pregnancy and application thereof.
Background
Abortion is a common problem and affects 25% of pregnant women under age 39. Miscarriage has been counted to affect approximately one third of pregnant women, and occurs most frequently in the mid-and early-term pregnancies. In recent years, early abortion has been increasingly developed year by year, and although early abortion prevention has been effective with the development of early intervention methods and drug therapy, the lack of early abortion prediction and diagnosis methods makes it difficult to effectively prevent and control the pregnancy diseases.
Normal communication between the mother and fetus is the basis for maintaining a successful and healthy pregnancy. The endometrium interstitial cells (ESCs) are continuously proliferated and differentiated to reach the decidualized state in the early pregnancy. Disruption of decidua function of ESCs prevents trophoblast cells from invading the endometrium, which can lead to pregnancy related diseases including infertility, early abortion, intrauterine late development and preeclampsia. HOXA10 and IL-6 have been shown to regulate ESCs cell proliferation and deciduation. In addition, steroid hormones and adhesion molecules are also involved in the regulation of cell proliferation and deciduation of ESCs and further affect embryo implantation and early pregnancy. However, the molecular mechanism leading to the defect of decidualization and its role in early abortion are still unclear at present.
Micrornas (mirnas) are a class of small, non-coding RNAs that can negatively regulate gene expression by binding to the 3' -UTR of a targeted mRNA. It was found that there is differential expression of mirnas in the endometrium during the decidualization process and the window phase of embryo implantation. miR-98 has been shown to regulate the proliferation and apoptosis of ESCs by targeting Bcl-xl in rat uterus. A recent study showed that miR-24 is down-regulated in decidua tissue of miscarriage patients and affects pregnancy outcome by regulating proliferation and apoptosis of ESCs. However, there are still many unknown mirnas involved in regulating the decidualization process of ESCs. miR-29a has been reported to induce apoptosis of trophoblast cells and to regulate trophoblast cell function in abortion and placental hyperplasia. Another study showed that miR-29a promotes granulosa cell proliferation by targeting aromatase expression and estradiol biosynthesis in polycystic ovary syndrome. In addition, miR-29a-5p has been shown to inhibit endometrial cancer cell proliferation by targeting TPX 2. Notably, miR-29a can inhibit apoptosis of ESCs by targeting apoptosis factors and participate in regulating the embryo implantation process. However, the role of miR-29a in affecting the proliferation and decidualization of ESCs and its potential application as a molecular marker for early abortion remains to be elucidated.
At present, no effective molecular marker exists in early abortion, and the treatment after abortion is not satisfactory due to the defects of the existing treatment and intervention technology, the miR-29 a-containing early abortion gene disclosed by the invention has the functions of regulating the proliferation and the decidualization of ESCs, evaluating the expression level of miR-29a in different gestation periods and early abortion and evaluating the diagnostic value of miR-29a applied to early abortion.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a miRNA biomarker for early abortion diagnosis of pregnancy and application thereof.
In the invention, the miRNA biomarker is hsa-miR-29a-3p (MIMAT 0000086).
The sequence of the hsa-miR-29a-3p is as follows: UAGCACCAUCUGAAAUCGGUUA is added.
Preferably, the hsa-miR-29a-3p is an decidua tissue miRNA biomarker.
The decidua tissue miRNA biomarker can be used for detecting early abortion of pregnancy and monitoring different pregnancy periods.
The invention provides application of the decidua tissue miRNA biomarker in screening or preparing a medicament for diagnosing early abortion.
The invention provides a group of miRNA primers for early abortion diagnosis, wherein the miRNA primers are hsa-miR-29a-3p primers.
The primer sequence of the hsa-miR-29a-3p is as follows: miR-29a-3p forward primer: 5'-TGCGCTAGCACCATCTGAAATC-3' and reverse primer: 5'-CCAGTGCAGGGTCCGAGGTATT-3'.
Preferably, the miRNA marker, the primer and the combination are applied to treatment of early abortion.
The purpose of the invention is realized according to the following technical scheme:
the method systematically researches the expression difference of the miR-29a of decidua tissues of different gestational periods and patients with abortion. The endometrium in the secretory phase is found to be remarkably up-regulated compared with the endometrial tissue miR-29a in the proliferative phase. Compared with normal early pregnancy, the expression of the early abortion endometrial periostracum tissue miR-29a is remarkably reduced. Consistent with clinical tissue samples, cell experiments prove that the miR-29a disclosed by the invention is up-regulated in the expression of ESCs decidualization models. While the down-regulation of miR-29a remarkably inhibits the cell proliferation of deciduated ESCs and the expression levels of deciduated markers PRL and IGFBP 1. The method can effectively predict different pregnancy periods and early abortion. The research result provides a basis for the prediction and diagnosis of early abortion and lays a foundation for the clinical application of early abortion molecular therapy.
Drawings
FIG. 1 shows the expression difference of miR-29a of endometrial or decidual tissue in different periods.
FIG. 2 shows the expression changes of miR-29a in deciduated ESCs.
FIG. 3 is a graph showing the cell proliferation effect of miR-29a down-regulation on deciduated ESCs.
FIGS. 4A-B are graphs showing the effect of miR-29a down-regulation on protein levels and relative quantification of decidua markers PRL and IGFBP1 of deciduated ESCs.
Detailed description of the preferred embodiments
In order that the invention may be more readily understood, specific embodiments thereof will be described further below.
Example 1: qRT-PCR detection of expression level of miR-29a of decidua tissue of endometrium and early-stage abortion patients in different pregnancy periods
(1) RNA extraction:
a portion of the tissue was cut with a surgical scissors and placed in a homogenizer tube containing 1mL of Trizol, and after thoroughly grinding using an electric homogenizer (ice-on operation), incubated for 5min, at 12000rpm, and centrifuged for 10 min. The supernatant was aspirated into a new 1.5mL centrifuge tube, 200. mu.L of chloroform was added thereto, shaken, allowed to stand at room temperature for 2min, at 4 ℃ and 12000rpm, and centrifuged for 10 min. The supernatant was aspirated into a new 1.5mL centrifuge tube, 600. mu.L of isopropanol was added, mixed well, left to stand at room temperature for 15min, 4 ℃, 12000rpm, centrifuged for 15min, and the supernatant was discarded. The precipitate was rinsed by adding 1mL of 75% absolute ethanol (750. mu.L absolute ethanol and 250. mu.L of water with LDEPC), centrifuged at 12000rpm for 5min at 4 ℃ and the supernatant discarded. Adding 1mL of anhydrous ethanol, rinsing the precipitate, centrifuging at 4 deg.C and 12000rpm for 5min, discarding the supernatant, and drying at room temperature for 10 min. 40 μ L of DEPC water was added to dissolve the RNA, and the RNA was stored in a freezer at-80 ℃ for further use.
(2) Reverse transcription reaction:
reverse transcription reaction conditions: 5min at 25 ℃, 15min at 50 ℃, 5min at 85 ℃ and 10min at 4 ℃; the reaction system is shown in Table 1.
TABLE 1 reverse transcription reaction System
(3) qPCR reaction:
the cDNA was diluted 7-fold and reacted under the following reaction conditions: 2min at 50 ℃ and 10min at 95 ℃; 30sec at 95 ℃, 30sec at 60 ℃, 40 cycles; the dissolution curves were plotted and the final data were expressed as 2-△△CtAnd (6) carrying out analysis. The reaction system is shown in Table 2.
TABLE 2 real-time fluorescent quantitative PCR reaction System
The results are shown in figure 1, the expression level of miR-29a in decidua tissue in early pregnancy is highest, the expression level of miR-29a in decidua tissue in early abortion is the next, the expression level of miR-29a in endometrial tissue in the secretory phase of menstrual cycle is the next, and the expression level of miR-29a in endometrial tissue in the proliferative phase of menstrual cycle is the lowest. The endometrium in the secretory phase is obviously up-regulated compared with the endometrium miR-29a in the proliferative phase. Compared with normal early pregnancy, the expression of decidua tissue miR-29a in early abortion is obviously reduced.
Example 2: isolated culture and decidualization model induction of human primary ESCs
Taking human uterine tissue, moving the human uterine tissue into a super-clean workbench, and putting the human uterine tissue into PBS containing double antibodies; carefully cleaning the peripheral redundant tissues, cleaning blood stains, gently scraping surface mucous membranes, separating endometrial tissues,cut the tissue into about 1mm3And (3) crushing the blocks. Washing tissue with PBS once, collecting tissue in a centrifuge tube, adding digestive juice, digesting in water bath at 37 deg.C by shaking table, adding complete culture medium after 40min to stop digestion, repeatedly blowing digestive juice with a suction tube, centrifuging the digestive juice at 300g for 5min, discarding supernatant, and retaining cell precipitate.
(1) Cell culture:
resuspending cell sediment by using a human ESCs complete culture medium, inoculating the cell sediment into a culture dish coated with polylysine in advance, performing static culture in a constant-temperature incubator at 37 ℃ and 5% CO2, changing the culture solution for the first time after 48 hours, changing the culture solution for 1 time every 3 days, and standing the cells for later use.
(2) Inducing in vitro molting:
and (5) carrying out in-vitro molting when the ESCs grow to about 60%. Primary human endometrial stromal cells were cultured in medium containing 2% FBS, estradiol E2(10nM), megestrol P4(1 μ M), cAMP (0.5mM) for 5 days to induce deciduation, and miR-29a inhibitor was treated 24h before induction. Morphological signs of molting: the form of the fibrillar spindle-shaped endometrium interstitial cells is changed into large, round and irregularly-shaped decidua cells; expression of deciduate molecular markers PRL and IGFBP-1.
Example 3: CCK-8 detects influence of miR-29a inhibitor on cell proliferation of deciduation-induced ESCs (endothelial cell proliferation)
(1) Cell culture:
the cells were cultured in complete medium containing 10% fetal bovine serum and 1% diabatic (100U/ml) human ESCs in an incubator containing 5% CO2 at 37 ℃ and saturated humidity. Changing the culture medium for 1 time at 2-3 days, and digesting and passaging with 0.25% pancreatin at 5-6 days.
(2) Grouping experiments:
a first control group;
deciduate group;
③ decidualization and inhibitor control group;
and fourthly, an ecdysis + miR-29a inhibitor group.
And (5) carrying out in-vitro molting when the ESCs grow to about 60%. Primary human endometrial stromal cells were cultured in medium containing 2% FBS, estradiol E2(10nM), megestrol P4(1 μ M), cAMP (0.5mM) for 5 days to induce deciduation, and miR-29a inhibitor was treated 24h before induction.
(3) And (3) detecting cell proliferation:
taking cells in logarithmic growth phase and good growth state, collecting the cells by trypsinization, adjusting the cell density to 5 multiplied by 104/mL by using a culture medium, inoculating the cells into a 96-well plate, setting a blank group (100 mu L of sterile PBS is added into holes around cell holes), and culturing overnight in a 5% CO2 incubator at 37 ℃ while setting 100 mu L of cell suspension per hole; after the cells adhere to the wall, the cells are cultured for 5 days in groups in an incubator at 37 ℃, 10 mu LCCK-8 is added into each hole, and the cells are cultured for 4 hours at 37 ℃; and measuring the light absorption value OD 450 of each hole by using a microplate reader.
As shown in fig. 3, endometrial stromal cell proliferation activity was increased after decidualization compared to the control group; the inhibitor control group has no influence on the proliferation activity of the endometrial stromal cells induced by the deciduzation basically, and the miR-29a inhibitor obviously inhibits the proliferation activity of the endometrial stromal cells in an deciduzation induced state (p is less than 0.01). The result shows that the reduction of the miR-29a level obviously inhibits the increase of endometrial stromal cell proliferation activity induced by decidualization.
Claims (6)
1. An miRNA marker for early abortion diagnosis, wherein the miRNA marker is hsa-miR-29a-3 p.
2. The miRNA marker of claim 1, wherein: the miRNA marker is an decidua tissue miRNA marker.
3. Use of the miRNA marker of any one of claims 1-2 in screening or preparing a medicament for diagnosing early abortion.
4. An miRNA primer used for early abortion diagnosis, wherein the miRNA primer is an hsa-miR-29a-3p primer.
5. A miRNA primer according to claim 4, for use in the prediction/diagnosis of early stage abortion.
6. Use of the miRNA markers, primers, and combinations of any one of claims 1-5 for the treatment of early stage miscarriage.
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| CN109423514A (en) * | 2017-08-24 | 2019-03-05 | 上海市计划生育科学研究所 | Recurrent spontaneous abortion related microRNA and its application |
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|---|---|---|---|---|
| WO2011029903A1 (en) * | 2009-09-10 | 2011-03-17 | Flemming Velin | Method for the preparation of micro-rna and its therapeutic application |
| CN109423514A (en) * | 2017-08-24 | 2019-03-05 | 上海市计划生育科学研究所 | Recurrent spontaneous abortion related microRNA and its application |
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| Title |
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| AIXIA LIU等: ""Loss of miR-29a impairs decidualization of endometrial stromal cells by TET3 mediated demethylation of Col1A1 promoter"", 《ISCIENCE》 * |
| HONG-FEI XIA等: ""MicroRNA expression and regulation in the uterus during embryo implantation in rat"", 《FEBS J》 * |
| QIAN YANG等: ""Association of the peripheral blood levels of circulating microRNAs with both recurrent miscarriage and the outcomes of embryo transfer in an in vitro fertilization process"", 《J TRANSL MED》 * |
| 张鹏等: ""miR-29a促进小鼠子宫内膜的蜕膜化"", 《中山大学学报(医学科学版)》 * |
| 张鹏等: ""抑制miR-29a表达对体外蜕膜化人子宫内膜基质细胞蜕膜催乳素分泌的影响"", 《山东医药》 * |
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