CN114622009A - miRNA molecular marker for early diagnosis of gestational diabetes and application thereof - Google Patents
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Abstract
The invention belongs to the technical field of gestational diabetes diagnosis marker molecules, and particularly relates to a miRNA (micro ribonucleic acid) molecular marker for early diagnosis of gestational diabetes and application thereof. The miRNA molecular marker comprises one or two combinations of miRNA-372 marker and miRNA-22 marker; the nucleotide sequence of the miRNA-372 marker is shown in a sequence table SEQID NO. 1; the nucleotide sequence of the miRNA-22 marker is shown in a sequence table SEQ ID NO. 2; the miRNA molecular marker is used for early diagnosis of gestational diabetes and is used for preparing a kit for early diagnosis of gestational diabetes.
Description
Technical Field
The invention belongs to the technical field of gestational diabetes diagnosis marker molecules, and particularly relates to a miRNA molecular marker for early diagnosis of gestational diabetes and application thereof.
Background
Gestational Diabetes (GDM) is defined as any degree of insulin resistance and glucose intolerance, developed or first discovered during pregnancy. GDM is one of the most common complications of pregnancy, with prevalence varying from 4% to 15%. GDM patients are at increased risk of developing a variety of chronic diseases after childbirth, including type 2 diabetes, independently of other clinical risk factors. In addition, GDM pregnant women are more prone to have adverse pregnancy events such as excessive amniotic fluid, difficult parturition, dead fetus, stillbirth and giant infants, and the incidence rate of GDM infant neonatal diseases is higher. GDM and its associated metabolic disorders are rapidly spreading worldwide, posing health and life risks to pregnant women and their newborns, which may persist into adulthood. However, the pathogenesis of GDM is unclear, most women have no diabetes or only mild symptoms before pregnancy, and aggravated diabetes only occurs during pregnancy, but once the woman is born, blood glucose levels and other physiological changes return to normal, which is the main clinical feature of GDM. This suggests that the placenta, which appears transiently during pregnancy, is the major effector organ of this condition. During pregnancy, the hormone levels of glucocorticoid, prolactin, progesterone, placental growth hormone and the like are increased, and the generation of insulin resistance and GDM can be further promoted.
On the placental tissues of pregnant women, the key role of the PI3K/AKT signaling pathway in insulin resistance is frequently studied in the analysis of the pathogenesis of diabetes. Glucose transporters (GLUTs) are an important component of the PI3K/AKT pathway, and the GLUT proteins-GLUT 1-5 and GLUT7 have been identified to date in 6, where GLUT4 is a key glucose transporter into skeletal muscle. GLUT4, encoded by SLC2A4 (chromosome 17p13), is translocated from the intercellular compartment to the cell membrane surface upon stimulation by insulin signals, and plays an important role in glucose homeostasis and in the pathogenesis of diabetes. Thus, the above observations suggest that placenta may also be a target for the PI3K/AKT/GLUT4 signaling pathway. GDM has great harm to puerpera and infants, but the current clinical diagnosis method is very limited, mainly including fasting blood glucose detection and oral glucose tolerance OGTT experiments, which are all diagnosis experiments detected after GDM occurs, and the development of early screening for GDM risk prediction and molecular markers for early disease diagnosis is urgently needed in clinic.
MicroRNAs (miRNAs) consist of a class of small noncoding RNA molecules 19-24 nucleotides long. They are highly conserved, but have a broad expression profile and are involved in many biological processes by regulating target gene expression at the post-transcriptional level. Several studies have shown that miRNAs are involved in the pathogenesis of type II diabetes, however, it is unclear which miRNAs exert regulatory and major functions in insulin pathway-mediated GDM, and the solution of these problems is helpful to develop molecular diagnostic products for early diagnosis of GDM.
Disclosure of Invention
In view of the above problems, the present invention aims to provide a miRNA molecular marker for early diagnosis of gestational diabetes and applications thereof, wherein the miRNA-372 marker can be used for early diagnosis of GDM and as a target for treatment of GDM.
The technical content of the invention is as follows:
the invention provides a miRNA molecular marker for early diagnosis of gestational diabetes, which comprises one or two combinations of miRNA-372 markers and miRNA-22 markers;
the nucleotide sequence of the miRNA-372 marker is shown in a sequence table SEQ ID NO. 1;
the nucleotide sequence of the miRNA-22 marker is shown in a sequence table SEQ ID NO. 2;
the invention also provides a mimic of the miRNA-372 marker, which is characterized in that the nucleotide sequence of a forward primer is shown as SEQ ID NO.3 of the sequence table, and the nucleotide sequence of a reverse primer is shown as SEQ ID NO.4 of the sequence table.
The invention also provides a mimic of the miRNA-22 marker, wherein the nucleotide sequence of the forward primer is shown as the sequence table SEQ ID NO.5, and the nucleotide sequence of the reverse primer is shown as the sequence table SEQ ID NO. 6.
The invention also provides an application of the miRNA-372 marker in preparing a gestational diabetes diagnosis kit;
the kit comprises the forward primer, the reverse primer molecule, and enzyme and reagent which are commonly used in PCR reaction.
The invention also provides an application of the miRNA-22 marker in preparing a gestational diabetes diagnosis kit;
the kit comprises the forward primer, the reverse primer molecule, and enzyme and reagent which are commonly used in PCR reaction.
The invention also provides a target gene SLC2A4 of the miRNA molecular marker for early diagnosis of gestational diabetes, the nucleotide sequence of which is shown in the sequence table SEQ ID NO. 20.
The invention has the following beneficial effects:
the miRNA molecular marker comprises one or two combinations of miRNA-372 markers and miRNA-22 markers, is used for early diagnosis of gestational diabetes and preparation of a kit, and can be used as a target spot for treating GDM; compared with cells cultured under normal glucose conditions, HTR8/SVneo cells cultured with high sugar showed lower expression of miR-372 and miRNA-22, indicating that they can both act as regulators of insulin signaling pathway. Western blot results show that the expression of GLUT4 in high-sugar culture cells transfected with miR-372 or miRNA-22 mimics is remarkably increased, and the expression of GLUT4 in cells transfected with miR-372 or miRNA-22 inhibitors is opposite. In cells treated with miR-372 or a mimic or inhibitor of miRNA-22, expression of PI3K, AKT, and IRS were all significantly reduced. GLUT4 was suggested to be the primary target of miR-372 in the insulin pathway. When the expression level of GLUT4 is increased, the uptake and utilization of glucose can be promoted. Due to the obvious positive regulation effect of miR-372 and miRNA-22 on GLUT4, the accumulation of glucose in GDM can be reduced, hyperglycemia is improved, the potential of the miR-372 as a GDM treatment target is highlighted, the down regulation of miR-22 and miR-372 expression can promote insulin resistance of GDM by regulating a PI3K/GLUT4 pathway, the increase of miR-22 and miR-372 expression can resist the influences, and miR-22 and miR-372 directly target 3' utr of SLC2A4 and regulate the expression of the miR-22 and miR-372.
Drawings
FIG. 1 is a graph showing the results of expression levels of miR-372 in placental tissue;
FIG. 2 is a graph showing the results of miR-372 expression level in HRT8/SVneo (human choriotrophoblast) cells;
FIG. 3 is a graph showing the results of IRS/PI3K/AKT expression of the insulin signaling pathway in placental tissue;
FIG. 4 is a graph showing the expression result of the regulation effect of IRS/PI3K/AKT on the miR-372 induced insulin signaling pathway;
FIG. 5 is a dual luciferase gene report.
Detailed Description
The present invention is described in further detail in the following description of specific embodiments and the accompanying drawings, it is to be understood that these embodiments are merely illustrative of the present invention and are not intended to limit the scope of the invention, which is defined by the appended claims, and modifications thereof by those skilled in the art after reading this disclosure that are equivalent to the above described embodiments.
All the raw materials and reagents of the invention are conventional market raw materials and reagents unless otherwise specified.
Example 1
Validation of miRNA markers for early diagnosis of gestational diabetes:
150 pregnant woman volunteers were selected, including 75 GDM pregnant women (GDM group) and 75 healthy pregnant woman control group (HC group).
1. Obtaining placental tissue
Placenta tissue was obtained within 15 minutes after delivery, placental lobules (cotyledons) were removed from the central region of the placenta, and the tissue pieces were cut into 1cm3The substrate plate and chorion surface on the cotyledons were removed and the villous tissue was collected, dissected to remove visible connective tissue and calcium deposits, blotted dry on filter paper, immediately snap frozen in liquid nitrogen and stored at-80 ℃ until further analysis.
2. Cell culture
HTR8/SVneo cell (Synbiotic Biotech, Inc. in China) is a human chorionic trophoblast cell, which is placed in RPMI-1640 medium containing 10% fetal bovine serum, 100. mu.g/mL penicillin and 100. mu.g/mL streptomycin at 37 ℃ with 5% CO2Culturing under the condition, and replacing the culture medium every day. Cells were washed twice with PBS and then at 3 × 10 per well5The density of individual cells was seeded into 6-well cell culture plates. The cells were divided into normal glucose group (5.57mmol/L) and three high glucose groups, 10.57mmol/L glucose (HGL group), 15.57mmol/L glucose (HG2 group) and 40.57mmol/L glucose (HG3 group), respectively, at 37 ℃ and 5% CO2And culturing for 24 hours.
Extraction of RNA and preparation of miRNAs
Using TRIzol reagent (magenta Biotechnology Co., Guangzhou, China) from 140mg placental tissue or 5X 10 according to manufacturer's protocol6Total RNA was extracted from individual cells, the quantity and quality of the total RNA obtained was evaluated using a spectrophotometer (NanoDrop, USA), small RNA molecules less than 35 nucleotides in length were defined as miRNA, and RNAMisi test was usedThe kit (Aidlab, Beijing, China) was isolated from the extracted total RNA.
RT-qPCR assay
Total RNA from all placental tissues and cell models was reverse transcribed using the TaKaRa PrimeScript II first strand cDNA synthesis kit (TaKaRa, japan). qPCR was performed on a CFX96 Touch real-time PCR detection system (Bio-Rad, USA) using the MiScript SYBR Green PCR Kit (TaKaRa, Japan). U6 snorRNA was used as an endogenous control, miRNA expression was normalized, and the relative expression of miR-372 and miR-22 target genes was measured using the 2- Δ Δ Ct method.
The primer sequences used were as follows:
hsa-miR-372-3p(SEQ IN NO.1)：
AAAGUGCUGCGACAUUUGAGCGU；
hsa-miR-22-3p(SEQ IN NO.2)：
AAAGUGCUGCGACAUUUGAGCGU；
the results of the experiments from which the cell samples were derived represent three independent experiments.
5. Transfection of miR-372 or miR-22 mimetics or antisense oligonucleotides
miR-372 and miR-22 inhibitors and mimetics were synthesized by Shanghai Integrated Biotechnology, Inc. (SIBS Co.). The sequences of the mimetics, inhibitors and controls are shown in table 1.
One day prior to transfection, HTR8/SVneo cells were plated at 2X 104Perwell was seeded in 24-well microplates with a glucose concentration of 50 mM. Lipofectamine RNAiMAX (Thermo Fisher Scientific, USA) was prepared according to the manufacturer's instructions. miR-372 mimetic/inhibitor/Negative Control (NC) was diluted in 200 μ L Opti-MEM serum-free medium and added to cells at a final concentration of 50nM miRNA mimetic and 100nM inhibitor/NC. Cells were incubated at 37 ℃ with 5% CO2Culturing for 24h under the condition.
TABLE 1 sequences of mimetics, inhibitors, controls
In the above inhibitor sequences, X in the inhibitor may be any one of A, C, G, U;
the hsa-miR-372-3p inhibitor comprises one of the following components:
SEQ ID NO.10：5'-ACGCUCUGAAGUCCAGCGACUUU-3'；
SEQ ID NO.11：5'-ACGCUCAAAUGUCGCAGCACUUU-3'；
SEQ ID NO.12：5'-ACGCGUCCAAGUACGUAGCCUUU-3'；
SEQ ID NO.13：5'-ACGCCUGGAGGAUUGACUGCUUU-3'；
SEQ ID NO.14：5'-ACGCCUGAUGGUUACAAUGCUUU-3'。
the sequence of the hsa-miR-22-3p inhibitor comprises the following one:
SEQ ID NO.15：5'-ACAGAAGUCCGUAGCUGCGCUU-3'；
SEQ ID NO.16：5'-ACAGUUCUUCAACUGGCAGCUU-3'；
SEQ ID NO.17：5'-ACAGAGUUUGCACAAGUCACUU-3'；
SEQ ID NO.18：5'-ACAGGUUUGAAGGUCCGUGCUU-3'；
SEQ ID NO.19：5'-ACAGUUAGGUUUGAACGAGCUU-3'。
6. immunoblotting
In the Western Blot (WB) of placental tissue, tissue proteins were detected by T-PERTMTissue protein extraction reagent (Thermo Fisher Scientific, usa) was used for extraction as described, and for WB experiments on cell samples, cells were harvested by centrifugation at 3000rpm for 5 minutes after transfection;
by M-PERTMMammalian protein extraction reagent (Thermo Fisher Scientific, USA) extracts total protein, protein concentration was identified using BCA protein assay kit (Beyotime Biotechnology, Shanghai, China) as per the instructions, equal amounts of protein (10 μ g, 15 μ L) were electrophoresed on 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE), 80V 0.5h, 120V 1h, electrotransferred to PVDF membrane (Millipop, USA) at 80mA for 3 hours, membrane was incubated with an antibody (antihuman) targeting IRS (1:500, Abcam, USA), PI3K (1:500, Abcam, USA), AKT (1:400, Abcam, USA), GLUT4(1:500, Abcam, USA) and β -actin (1:1000, Abcam, USA) at 4 ℃Overnight. After washing with TBST, the membrane was incubated with the corresponding horseradish peroxidase-labeled secondary antibody (goat-anti-rabbit, 1: 10000, Abcam, USA) for 2 hours;
the luminescence was dropped on a film, placed into a chemiluminescence apparatus iBright FL1500 (Invitrogen co., USA, Invitrogen co., USA) for automatic exposure imaging, and the optical density of the target protein was quantified using a gel image processing system, and the experiment was repeated at least 3 times under the same conditions with β -actin as an internal control.
7. Dual luciferase gene reporter detection
miRNA-372 and miRNA-22 target genes are predicted and analyzed by using an online tool RNA22 v2 microRNA target detection (https:// cm. jefferson. edu/RNA 22/Interactive). Through database query, the SLC2A4 gene (also called GLUT4) in the insulin pathway is predicted to be regulated by miR-372 and miR-22. The tool predicts that miR-372 has two binding sites in the 3' UTR of SLC2a 4: the miR-22 could target the 333-345bp of SLC2A 43' UTR, and the miR-372 could target the 68-74bp (SEQ ID NO. 20AGCACUU).
Based on the binding sites, SIBS designed and synthesized wild-type and mutant plasmids, and the plasmid vector was psiCHECK-2 vector, and the sequence information is shown in Table 2. The reporter plasmid was co-transfected with miR-372 or miR-22 mimetics or miR-372 or miR-22NC and HTR8/SVneo cells using Lipofectamine 2000. After 48 hours, luciferase activity was assessed using the dual luciferase reporter assay system (Promega Corporation, USA) according to the manufacturer's instructions.
Results are representative from at least three independent experiments, each sample having three parallel wells.
TABLE 2 plasmid construction information
The above test results are as follows:
data shown are expressed as means ± Standard Deviation (SD). Comparisons were calculated using either a t-test between groups or a one-way analysis of variance (ANOVA) (> two groups) between groups, respectively, where a p-value <0.05 was considered statistically significant.
Data analysis employed GraphPad Prism 8.0.2 software (GraphPad software, La Jolla, Calif.) or SPSS 21.0 software (IBM).
1) Expression of miR-372 and miR-22 in placental tissue
qPCR detection is carried out on the placenta tissues from the GDM group and the HC group, and the results are shown in figure 1, and the expression of miR-372 and miR-22 in the placenta tissues of the GDM group participants is obviously lower than that in the HC group (P < 0.001).
Wherein the variation difference of the two miRNAs is large, the variation of the miR-22 is the largest, and the difference is larger than 5000 times.
2) Expression of miR-372 and miR-22 in HRT8/SVneo cells
As shown in FIG. 2, HRT8/SVneo cells were stimulated by high glucose gradients (10, 30 and 50mM) by establishing the HRT8/SVneo glucose tolerance cell model (FIG. 2A). Expression of miR-372 in HRT8/SVneo cells was measured and it was found that expression of both miRNAs gradually decreased with increasing glucose concentration (FIG. 2B), which is consistent with the results for GDM and HC tissues, indicating that high sugar treatment is negatively associated with miR-372 and miR-22 expression.
3) Expression of PI3K/AKT/IRS in the placental insulin signaling pathway
As shown in figure 3, the expression of PI3K, AKT, IRS and GLUT4 in the placenta of female in the GDM group is obviously reduced compared with that in the HC group, the trend is consistent with the change of miR-372 expression in placenta tissues, the result shows that insulin resistance can inhibit a PI3K/AKT/IRS pathway, and miR-372 and miR-22 can regulate the cell pathway.
4) miR-372 and miR-22 regulate PI3K/AKT/IRS branch of insulin signaling pathway
Further researching the relation between the change of insulin signal pathway protein and the expression difference of two miRNAs in GDM, and discussing the regulation and control effect of miR-372 and miR-22 on PI3K/AKT/IRS pathway protein in HRT8/SVneo cells transfected with miR-372 or miR-22 mimics or inhibitors. As shown in FIG. 4, the results show that GLUT4 expression is significantly increased in high sugar cultured HRT8/SVneo cells transfected with miR-372 or miR-22 mimics; in contrast, expression of GLUT4 was significantly reduced in miR-372 or miR-22 inhibitor treated cells. Meanwhile, in cells treated with miR-372 or miR-22 mimics or inhibitors, the expression of PI3K, AKT and IRS is significantly reduced.
Wherein, the regulation effect of miR-372 on GLUT4 expression is stronger than that of miR-22, and the regulation effect of miR-22 on PI3K, AKT and IRS expression is stronger than that of miR-372.
5) SLC2A4 is the target gene of miR-372 and miR-22
To further verify whether miR-372 and miR-22 interact directly with genes involved in the insulin signaling pathway, a dual luciferase gene reporter assay was established and the correlation between these mirna and their putative target genes was evaluated. By querying the database (using the RNA22 web tool), one could predict a gene that functions in the insulin signaling pathway SLC2a4 (the gene encoding GLUT4), which is clearly regulated by miR-372. Thus, plasmids containing either wild-type or mutant putative miR-372 targeting the 3' UTR of SLC2a4 were constructed. As shown in FIG. 5, dual luciferase assay results show that miR-22 is significantly reduced, while miR-372 increases wild-type SLC2A 43' UTR-driven luciferase activity. However, neither the AKT 13 ' UTR-driven luciferase activity nor their respective mutated 3' UTR-driven luciferase activity were significantly affected, suggesting that miR-372 and miR-22 directly target and regulate expression of the 3' UTR of SLC2a 4.
Example 2
Gestational diabetes diagnosis kit:
TABLE 3 diagnostic kit containing hsa-miR-372-3p marker
Most of the above reagents have been commercialized and supplied by supplier companies.
Example 3
Gestational diabetes diagnosis kit:
TABLE 4 diagnostic kits containing hsa-miR-22-3p markers
Most of the above reagents have been commercialized and supplied by supplier companies.
The serum of 5 diabetic pregnant women was selected and tested for accuracy and stability using the kits prepared in examples 2 and 3, with the following results.
Table 5 accuracy testing of the example 2 and example 3 kits
As can be seen from Table 5, compared with the control kit, the kit of the invention has a sample detection rate of 100% for the pregnant woman with diabetes, and the related performance of the control kit reaches R299, it can be seen that the sensitivity and accuracy of the kits described in examples 2 and 3 can meet the relevant requirements.
The two kits are respectively placed at room temperature (20-25 ℃) and low temperature (2-8 ℃) for a period of time, then the serum of the above 5 patients is continuously adopted for kit detection, and the results are shown in the following table.
Table 6 stability testing of the kits of example 2 and example 3
As can be seen from table 6, the detection signal values of the kits of embodiment 2 and embodiment 3 of the present invention at different times under low temperature storage are respectively counted, and the results show that the kits still have high detection stability even after being stored for 12 months.
Claims (8)
1. A miRNA molecular marker for early diagnosis of gestational diabetes mellitus, wherein the miRNA molecular marker comprises one or two combinations of miRNA-372 marker and miRNA-22 marker;
the nucleotide sequence of the miRNA-372 marker is shown in a sequence table SEQ ID NO. 1;
the nucleotide sequence of the miRNA-22 marker is shown in a sequence table SEQ ID NO. 2.
2. The mimic of the miRNA-372 marker of claim 1, wherein the nucleotide sequence of the forward primer is shown as SEQ ID No.3 of the sequence table, and the nucleotide sequence of the reverse primer is shown as SEQ ID No.4 of the sequence table.
3. The mimic of the miRNA-22 marker of claim 1, wherein the nucleotide sequence of the forward primer is shown in SEQ ID No.5 of the sequence table, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.6 of the sequence table.
4. Use of the miRNA-372 marker of claim 1 in the preparation of a gestational diabetes diagnosis kit.
5. A kit for early diagnosis of gestational diabetes by using miRNA molecular markers, which is characterized by comprising the forward primer, the reverse primer molecule and enzymes and reagents commonly used in PCR reaction of claim 2.
6. Use of the miRNA-22 marker of claim 1 in the preparation of a gestational diabetes diagnosis kit.
7. A kit for early diagnosis of gestational diabetes by using miRNA molecular markers, which is characterized by comprising the forward primer, the reverse primer molecule and enzymes and reagents commonly used in PCR reaction of claim 2.
8. A target gene SLC2A4 of miRNA molecular marker for early diagnosis of gestational diabetes.
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| CZ309886B6 (en) * | 2022-08-12 | 2024-01-10 | GeneSpector s.r.o. | A gestational diabetes mellitus prediction method according to the expression profile of cardiovascular miRNAs |
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