CN108642175B - Early embryo diapause villus tissue miRNAs biomarker and detection method of expression quantity thereof - Google Patents

Abstract

The invention discloses early embryo diapause villus tissue miRNAs biomarkers related to early embryo diapause degree, which comprise hsa-miR-136-5p, hsa-miR-1301-3p, hsa-miR-135b-5p, hsa-141-3p, hsa-miR-19b-3p and hsa-iR-486-5 p. Accordingly, the inventors have also designed corresponding reverse transcription or real-time fluorescent PCR primers targeting the above miRNAs biomarkers. The compounds can be used as biomarkers for detecting early embryo diapause by villus samples, or used for preparing tools such as diagnostic reagents or biochips, and the like, have important potential value and application prospect for diagnosis and treatment of early embryo diapause, can provide theoretical basis for the later research of diagnosing human early embryo diapause, tissues and serum miRNAs from a molecular level, and have important theoretical significance and potential practical value.

Description

Detection of miRNAs biomarkers and their expression levels in early embryonic aborted villous tissue

technical field

The invention belongs to the technical field of biomarkers in biomedicine, in particular to a method for detecting miRNAs biomarkers in early embryonic aborted villous tissue and their expression levels.

Background technique

Early Embryonic Arrest (EEA) is one of the main causes of female infertility. To date, the etiology of early embryonic cessation is unclear and treatment options are limited, seriously affecting the physical and mental health of women of childbearing age.

MicroRNAs (miRNAs) are an important regulatory molecule in modern biomedicine. They regulate various functions of all cells, tissues and organs in the human body through the regulation of gene transcription and expression, and play an important role in the occurrence and prevention of human diseases. important role. A large number of studies have found that miRNAs can widely and stably exist in extracellular fluids, including serum, plasma and interstitial fluid. They are not only regulatory molecules for intracellular gene transcription and expression, but also signaling molecules for information transmission between cells. During the pathological process, the expression profile of miRNAs in tissue fluid will have characteristic changes, which are helpful for the diagnosis and prognosis of the disease, and are even closely related to the clinical staging and grading.

Under normal functional state, the types of circulating miRNAs and their expression abundance may be in a relatively stable and balanced state. Different degrees of repeated injury process during the onset of early embryo arrest may directly or indirectly disrupt the homeostasis of miRNAs in villus tissue, manifesting as abnormal metabolism or a significant increase in the expression levels of miRNAs in certain tissues under specific disease states. high or low. So far, there are no published reports on miRNAs in early embryonic aborted villous tissue.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is to provide a detection method of miRNAs biomarkers in villous tissue of early embryonic abortion and their expression levels, so as to provide support for the accurate diagnosis of early embryonic abortion in clinic.

In order to solve the above-mentioned technical problem, the present invention adopts above-mentioned technical scheme:

Biomarkers of miRNAs in early embryonic arrested villus tissue, including hsa-miR-136-5p, hsa-miR-1301-3p, hsa-miR-135b-5p, hsa-141-3p, hsa-miR-19b-3p and hsa-iR-486-5p, which have the base sequences of SEQ.ID.No.1 to SEQ.ID.No.6 in the Sequence Listing, respectively.

The above-mentioned biomarkers of miRNAs in the embryonic aborted villous tissue are derived from human villous tissue.

The application of miRNAs biomarkers in the villous tissue of early embryonic abortion in the preparation of reagents, kits or biochips for diagnosing or assisting the diagnosis of early embryonic abortion.

The application of the above-mentioned early embryonic aborted villous tissue miRNAs biomarkers in the preparation or screening of drugs for the treatment of early embryonic aborted.

The primers designed to target the miRNAs biomarkers of the above-mentioned early embryo arrest villus tissue include the base sequences of SEQ.ID.No.7 to SEQ.ID.No.14 in the sequence listing.

The application of the above primers in the preparation of reagents, kits or biochips for diagnosing or assisting in the diagnosis of early embryonic infertility.

The application of the above primers in the preparation or screening of drugs for treating early embryonic abortion.

The method for detecting the expression of miRNAs biomarkers in the aborted villous tissue of the above-mentioned early embryo includes the following steps:

(1) Total RNA extraction

Take villus tissue, add liquid nitrogen and grind it into powder, take 50-100 mg of ground powder into a 2 mL centrifuge tube, and add 1 mL of TRIZOL to fully lyse it. After standing at room temperature for 5 minutes, add 200 μL of chloroform, and vortex to mix for 15 seconds. Let stand on ice for 15 minutes; centrifuge at 12,000g for 15 minutes at 4°C; aspirate the upper aqueous phase, add an equal volume of pre-cooled isopropanol, mix gently, and let stand on ice for 10 minutes; centrifuge at 12,000g at 4°C for 10 minutes, discard the Add 10 mL of pre-cooled 75% ethanol prepared with DEPC-treated water, carefully resuspend the pellet, centrifuge at 12,000g at 4°C for 10 minutes, discard the supernatant; repeat the washing of the pellet; aspirate the supernatant, and place it on a clean bench to air dry Precipitate for 5-10 minutes; finally add 20 μL of double distilled water treated with DEPC;

(2) Removal of genomic DNA from total RNA

The kit was used to remove genomic DNA. The reaction system included 0.4 μL 2U/μL DNase I, 2 μL 10* reaction buffer, 0.6 μL 20U/μL RiboLock RNase Inhibitior, and 4 μg total RNA was added to each reaction system, and the total reaction system was 20 μL; The conditions are: reaction at 37°C for 30 minutes, followed by inactivation at 75°C for 10 minutes;

(3) reverse transcription

The reverse transcription reaction of miRNAs was performed using a reverse transcription kit and miRNAs-specific stem-loop structure reverse transcription random primers; the reaction system included 0.5 μL reverse transcription random primers, 2 μL 5* reverse transcription buffer, 1 μL 10 mmol/L dNTP mixture, 0.5 μL μL 20U/μL RiboLock RNase Inhibitior, 0.5 μL 200U/μL Reverse Transcriptase, 1 μg RNA from which gene DNA had been removed was added to each reaction system, the total reaction system was 10 μL; the reaction conditions were incubated at 65°C for 5 minutes and immediately placed on ice for 5 minutes, and then React at 42°C for 60 minutes, and finally incubate at 70°C for 5 minutes;

(4) Real-time fluorescence quantitative PCR

5 μL 2* SYBR Green mixture, 0.05 μL ROX, 1.4 μL miRNAs primer mixture, 1 μL diluted cDNA, 2.55 μL double-distilled water, the total reaction system was 10 μL, and the detection of each miRNA was performed in 4 parallel replicates; using fast real-time fluorescence Quantitative PCR instrument was used for detection. The reaction conditions of the instrument were as follows: 95°C for 2 minutes, 95°C for 5 seconds, and 60°C for 30 seconds, for 40 cycles.

The reverse transcription random primer in step (3) has the base sequence of SEQ.ID.No.14 in the sequence table, and the primer mixture in step (4) includes the sequence table SEQ.ID.No.7 to SEQ.ID.No. 13 base sequence.

The inventors used high-throughput sequencing and real-time quantitative PCR methods for detection, and found that there are high- or low-expression miRNAs biomarkers in the early embryonic aborted villous tissue that are highly expressed or lowly expressed in the tissues of patients with early embryonic abortion and are related to the degree of early embryonic abortion. Including hsa-miR-136-5p, hsa-miR-1301-3p, hsa-miR-135b-5p, hsa-141-3p, hsa-miR-19b-3p and hsa-iR-486-5p, which have Sequence Listing Base sequences of SEQ.ID.No.1 to SEQ.ID.No.6. Accordingly, the inventors also designed corresponding reverse transcription or real-time fluorescent PCR primers targeting the above-mentioned miRNAs biomarkers, including the base sequences of SEQ.ID.No.7 to SEQ.ID.No.14 in the sequence listing. Experiments confirmed that hsa-miR-136-5p, hsa-miR-1301-3p, hsa-141-3p, hsa-miR-135b-5p were highly expressed in the villus tissue of early embryonic arrest patients, hsa-miR-19b -3p and hsa-miR-486-5p are lowly expressed in the villus tissue of patients with early embryonic arrest, therefore, they can be used as biomarkers for the detection of early embryonic arrest in villus samples, or for the preparation of diagnostic reagents or biochips, etc. It has important potential value and application prospects for the diagnosis and treatment of early embryonic abortion, and can provide a theoretical basis for the future research on the diagnosis of human early embryonic abortion, tissue and serum miRNAs from the molecular level. It has great theoretical significance and potential. practical value.

Description of drawings

Figure 1 is a histogram of the expression of has-miR-1301-3p in the control and experimental groups detected by qPCR.

Figure 2 is a histogram of the expression of has-miR-141-3p in the control and experimental groups detected by qPCR.

Figure 3 is a histogram of the expression of has-miR-19b-3p in the control and experimental groups detected by qPCR.

Figure 4 is a histogram of the expression of has-miR-135b-5p in the control group and the experimental group detected by qPCR.

Figure 5 is a histogram of the expression of has-miR-486-5p in the control and experimental groups detected by qPCR.

Figure 6 is a histogram of the expression of has-miR-136-5p in the control group and the experimental group detected by qPCR.

In the figure: the abscissa is the control group and the experimental group, and the ordinate is the average expression level.

Detailed ways

The present invention will be described in detail below through the examples and in conjunction with the accompanying drawings, and the conditions and methods that are not indicated in the examples are all carried out under normal conditions.

In this example, the early embryonic aborted villous tissue miRNAs biomarkers are used to detect early embryonic abortion, and the specific process is as follows:

1. Screening candidate early embryo arrest-related miRNAs by high-throughput sequencing of microRNAs:

The villus tissues of 3 normal early-pregnancy healthy controls and 3 patients with early embryo stoppage were collected, total RNA was extracted by Trizol method, and the obtained total RNA was subjected to quality inspection to ensure that the concentration and integrity of total RNA met the sequencing requirements . 1.5μg total RNA was taken from each sample, and the 18-32nt fragments were enriched with a small RNA enrichment kit. Small RNASample PrepKit was used to construct a small RNA library. The purified small RNAs were ligated with adapter primers using T4RNA ligase. To the 5' and 3' ends of the sequences of the two libraries, the first strand of cDNA was synthesized by reverse transcription, and the small RNA library was established by ordinary PCR method. The library was subjected to high-throughput sequencing using the Illumina HiSeq 2500 high-throughput sequencing platform. The small RNA sequence with a length of 51 nt obtained by sequencing was finally obtained by removing the linker, low-quality and contaminating sequences, and finally obtaining a high-quality small RNA sequence of 18-32 nt. The obtained small RNA sequences were compared with the reference genome, and then the matched sequences were compared with the sequences in the database, and the miRNA sequences were screened and classified and annotated, and their expression was analyzed. The expression of miRNAs in the sample was normalized to TPM (Tags per million), and the differences in the expression of known miRNAs in the two groups of samples were analyzed and counted, and the difference miRNAs were screened by using the P value and fold change value of the T test according to the preset conditions. , to preliminarily screen out the miRNAs specifically expressed in the villous tissue of patients with early embryonic arrest. The screening criteria are:

1) Fold change of expression level (Fold Change) ≥ 2 (low expression is < 0.5);

2) P value ≤ 0.01;

Thirty-six candidate miRNAs were screened, among which 22 miRNAs were up-regulated, namely: hsa-miR-549a, mir-200-x, mir-6236-y, mir-3940-y, hsa-miR-2276-3p , mir-8304-y, hsa-miR-4800-3p, mir-4661-x, hsa-miR-589-3p, hsa-miR-4664-5p, hsa-miR-556-3p, novel-m0066-5p , novel-m0045-5p, hsa-miR-136-5p, hsa-miR-3614-3p, hsa-miR-1301-3p, hsa-miR-24-1-5p, mir-24-x, hsa-miR -141-3p, hsa-miR-3614-5p, hsa-miR-2116-3p, hsa-miR-135b-5p; 14 miRNAs were down-regulated, respectively: mir-126-x, mir-4483-y , hsa-mir-33a-5p, mir-539-y, mir-17-x, hsa-miR-19b-3p, mir-19-y, mir-142-y, hsa-miR-144-5p, hsa -miR-486-5p, hsa-miR-16-1-3p, mir-144-x, mir-486-x, hsa-miR-144-rp.

2. Validation of miRNAs in villous tissue of early embryonic arrest patients by real-time quantitative PCR

Four miRNAs (hsa-miR-136-5p, hsa-miR-1301-3p, hsa-miR-135b-5p and hsa-141-3p) and two The down-regulated expression of miRNAs (hsa-miR-19b-3p, hsa-miR-486-5p) was used as a biomarker for the diagnosis of early embryo arrest, and the next step was real-time fluorescence quantitative PCR verification analysis. The villous tissues of 6 patients with early embryo arrest were collected as the experimental group, and 6 normal early-pregnancy healthy controls matched in age and sex were also collected (Table 1). The specific detection process is as follows:

(1) Extraction of total RNA: Take an appropriate amount of villus tissue and add it to liquid nitrogen and quickly grind it into powder. Take 50-100 mg of the ground powder and put it into a 2 mL centrifuge tube. At the same time, add 1 mL of TRIZOL to make it fully lysed. After standing at room temperature for 5 minutes, add 200 μL of chloroform, vortex for 15 seconds, and let stand on ice for 15 minutes; centrifuge at 12,000g at 4°C for 15 minutes; carefully aspirate the upper aqueous phase, add an equal volume of pre-cooled isopropanol, mix gently, and let stand on ice for 10 minutes ; Centrifuge at 12000g at 4°C for 10 minutes, discard the supernatant; add 10 mL of pre-cooled 75% ethanol prepared with DEPC-treated water, carefully resuspend the pellet, centrifuge at 12,000g at 4°C for 10 minutes, discard the supernatant; repeat the washing of the pellet; fully Aspirate the supernatant and place it on the ultra-clean workbench to air dry the pellet (5-10 minutes). The pellet should not be blown too dry to prevent the RNA from drying and not easily dissolved; finally, add 20 μL of double distilled water treated with DEPC.

(2) Removal of genomic DNA in total RNA: using a kit for removing genomic DNA, the reaction system includes 0.4 μL DNase I (2U/μL), 2 μL 10* reaction buffer, 0.6 μL RiboLock RNase Inhibitior (20U/μL) , 4 μg of total RNA was added to each reaction system, and the total reaction system was 20 μL. The reaction conditions of this method are: reaction at 37°C for 30 minutes, followed by inactivation at 75°C for 10 minutes.

(3) Reverse transcription: The reverse transcription reaction of miRNAs was performed using reverse transcription kits and miRNAs-specific stem-loop structure reverse transcription random primers. The reaction system includes 0.5μL reverse transcription random primer, 2μL 5* reverse transcription buffer, 1μL 10mmol/L dNTP mixture, 0.5μL RiboLock RNase Inhibitior (20U/μL), 0.5μL reverse transcriptase (200U/μL), each 1 μg of RNA from which gene DNA has been removed was added to the reaction system, and the total reaction system was 10 μL. The reaction conditions for this method were incubation at 65°C for 5 minutes and immediately placed on ice for 5 minutes, followed by a reaction at 42°C for 60 minutes, and a final incubation at 70°C for 5 minutes.

(4) Real-time quantitative PCR: 5μL 2* SYBR Green mixture, 0.05μL ROX (instrument fluorescence calibration solution), 1.4μL miRNAs primer mixture, 1μL diluted cDNA, 2.55μL double distilled water, the total reaction system is 10μL , the detection of each miRNA was performed in 4 parallel replicates. The ABI 7500 type rapid real-time fluorescence quantitative PCR instrument was used for detection. The reaction conditions of the instrument were: 95 °C for 2 minutes, 95 °C for 5 seconds, 60 °C for 30 seconds, and 40 cycles were performed to complete the early embryo arrest. Villous tissue miRNAs biological The expression levels of the markers were detected (see Table 3 for specific primers).

(5) In the real-time quantitative PCR test, the Ct value is used as the relative quantitative parameter of miRNAs in the results. The principle is that the Ct value is linearly inversely proportional to the logarithm of the copy number of the original template in the sample. Among them, "C" in the Ct value stands for Cycle, and "t" stands for detection threshold, which means the number of cycles required for the fluorescence signal intensity to reach the threshold during PCR amplification. In this test, when the Ct value is greater than 35, it is considered that the expression of the miRNA in the sample is low and the miRNA cannot be detected or does not contain the miRNA; if the amplification efficiency of the target miRNAs and the internal reference is the same, it can be calculated according to ΔCt=target gene Ct value - Ct value of the reference gene,

ΔΔCt = ΔCt value of each case sample - ΔCt average value of control group, ΔCt in this experiment = target miRNACt - internal reference Ct, so as to obtain the quantification of target miRNA relative to the internal reference. The 2 ^-ΔΔct method was used to calculate the fold change of the expression of miRNA in the experimental group relative to the control group. The experiment set up a negative control and repeated experiments, and all real-time quantitative PCR were repeated three times. The Mann-Whitney test method was used to calculate the differential expression levels of each miRNA between the two groups. Statistically, when P<0.05, the difference was considered statistically significant. The data processing results of differentially expressed miRNAs are expressed as mean + standard error, and a bar chart with error bars is drawn at the same time; the results are shown in Figure 1-Figure 6, hsa-miR-136-5p, hsa-miR-135b-5p and hsa-141-3p were highly expressed in the villous tissue of early embryonic arrest patients, hsa-miR-19b-3p and hsa-miR-486-5p were low in early embryonic arrest patients expression, consistent with the initial screening results.

Table 1 12 tissue samples

Table 2 Sequences of 6 miRNAs biomarkers for diagnosing early embryonic arrest

miRNA names sequence has-miR-136-5p acuccauuuguuuugaugaugga has-miR-1301-3p uugcagcugccugggagugacuuc has-miR-135b-5p uauggcuuuuucauuccuauguga has-miR-141-3p uaacacugucugguaaagaugg has-miR-19b-3p gugcaaauccaugcaaaacuga has-miR-486-5p uccuguacugagcugccccgag

Table 3 Real-time fluorescent quantitative PCR primer information

To sum up, the present invention establishes a screening method for tissue miRNAs biomarkers and an expression profile of miRNAs in the villous tissue of early embryonic arrest, and finds that miRNAs are abnormally expressed in the villous tissue of patients with early embryonic arrest, revealing that the miRNAs in the villous tissue have an impact on early embryonic arrest. value of reproductive testing. Among them, hsa-miR-136-5p, hsa-miR-1301-3p, hsa-141-3p, hsa-miR-135b-5p were highly expressed in the tissues of patients with early embryonic arrest, hsa-miR-19b-3p , hsa-miR-486-5p was lowly expressed in early embryonic abortion, which may be a characteristic miRNAs molecule in patients with early embryonic abortion, and can be used as a marker for diagnosis or auxiliary diagnosis and screening of drugs for early embryonic abortion. Among them, hsa-miR-136-5p, hsa-141-3p, hsa-miR-1301-3p and hsa-miR-135b-5p were up-regulated markers, hsa-miR-19b-3p and hsa-iR- 486-5p are down-regulated markers, which can be used as early embryo arrest diagnostic markers or auxiliary diagnostic markers in villous tissue samples. miRNAs can be used as diagnostic markers for early embryonic abortion and can be used for auxiliary diagnosis of early embryonic abortion patients. This has important academic significance and application prospects for the auxiliary diagnosis of early embryo arrest and enrichment of the pathological mechanism of early embryo arrest.

sequence listing

<110> The First Affiliated Hospital of Guangxi Medical University

Nanning Weierkai Biotechnology Co., Ltd.

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Claims (6)

1. The miRNAs biomarkers of early embryo shutdown villus tissues are characterized by comprising hsa-miR-136-5p, hsa-miR-1301-3p, hsa-miR-135b-5p, hsa-141-3p, hsa-miR-19b-3p and hsa-iR-486-5p which are respectively the base sequences of sequence tables SEQ ID No.1 to SEQ ID No. 6.

2. Use of the early embryo diapause villus tissue miRNAs biomarkers of claim 1 in the preparation of a reagent, kit or biochip for diagnosis or assisted diagnosis of early embryo diapause.

3. A primer designed with the early embryo diapause villus tissue miRNAs biomarker as described in claim 1, which is characterized by comprising the base sequence of SEQ ID No.7 to SEQ ID No. 14.

4. Use of the primer of claim 3 for the preparation of a reagent, a kit or a biochip for the diagnosis or the auxiliary diagnosis of early embryo diapause.

5. The method for the non-diagnostic detection of the expression level of early embryo diapause villus tissue miRNAs biomarkers of claim 1, comprising the steps of:

(1) total RNA extraction

Adding liquid nitrogen into villus tissue, grinding into powder, putting 50-100mg of the ground powder into a 2mL centrifuge tube, simultaneously adding 1mL TRIZOL to fully crack the villus tissue, standing at room temperature for 5 minutes, adding 200 mu L chloroform, mixing uniformly by vortex for 15 seconds, and standing on ice for 15 minutes; centrifuging at 12000g at 4 ℃ for 15 minutes; absorbing the upper-layer water phase, adding precooled iso-propanol, lightly mixing uniformly, and standing on ice for 10 minutes; centrifuging at 12000g at 4 ℃ for 10 minutes, and removing supernatant; adding 10mL of 75% ethanol prepared from precooled DEPC-treated water, carefully resuspending the precipitate, centrifuging at 12000g at 4 ℃ for 10 minutes, and discarding the supernatant; washing the precipitate repeatedly; sucking out the supernatant, and placing the supernatant on a super-clean workbench for air drying and precipitating for 5 to 10 minutes; finally, 20 μ L of double distilled water treated with DEPC was added;

(2) removal of genomic DNA from Total RNA

The method is carried out by adopting a kit for removing genome DNA, a reaction system comprises 0.4 mu L of 2U/. mu.L DNase I, 2 mu L of 10-inch reaction buffer solution and 0.6 mu L of 20U/. mu.L RiboLock RNase inhibitor, 4 mu g of total RNA is added into each reaction system, and the total reaction system is 20 mu L; the reaction conditions are as follows: reacting at 37 ℃ for 30 minutes, and then inactivating at 75 ℃ for 10 minutes;

(3) reverse transcription

Carrying out reverse transcription reaction on the miRNAs by adopting a reverse transcription kit and a miRNAs specific stem-loop structure reverse transcription random primer; wherein the reaction system comprises 0.5 muL of reverse transcription random primer, 2 muL of 5-prime reverse transcription buffer solution, 1 muL of 10mmol/L dNTP mixture, 0.5 muL of 20U/muL RiboLock RNase inhibitor and 0.5 muL of 200U/muL reverse transcriptase, 1 mug of RNA with gene DNA removed is added into each reaction system, and the total reaction system is 10 muL; the reaction conditions are that the mixture is incubated at 65 ℃ for 5 minutes and immediately placed on ice for 5 minutes, then reacted at 42 ℃ for 60 minutes, and finally incubated at 70 ℃ for 5 minutes;

(4) real-time fluorescent quantitative PCR

5 μ L of 2 SYBR Green mixed liquor, 0.05 μ L ROX, 1.4 μ L miRNAs primer mixed liquor, 1 μ L diluted cDNA, 2.55 μ L double distilled water, 10 μ L total reaction system, and 4 parallel repeats of each miRNA detection; the rapid real-time fluorescence quantitative PCR instrument is used for detection, and the reaction conditions of the instrument operation are as follows: 40 cycles of 95 ℃ for 2 minutes, 95 ℃ for 5 seconds, and 60 ℃ for 30 seconds were performed.

6. The method for detecting the expression level of miRNAs biomarkers in early embryo diapauzing villus tissue without diagnostic purpose as claimed in claim 5, wherein: the reverse transcription random primer in the step (3) is a base sequence of a sequence table SEQ ID No.14, and the primer mixed solution in the step (4) comprises base sequences of sequence tables SEQ ID No.7 to SEQ ID No. 13.

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