CN112626200B

CN112626200B - Application of serum microvesicle-asparagine endopeptidase pseudogene 1 in diagnosis and recurrence prediction of endometriosis

Info

Publication number: CN112626200B
Application number: CN202110033775.9A
Authority: CN
Inventors: 邱君君; 华克勤; 孙淑根; 郭靖靖; 郭晨妍; 瞿欣瑜
Original assignee: Obstetrics and Gynecology Hospital of Fudan University
Current assignee: Obstetrics and Gynecology Hospital of Fudan University
Priority date: 2021-01-12
Filing date: 2021-01-12
Publication date: 2022-07-22
Anticipated expiration: 2041-01-12
Also published as: CN112626200A

Abstract

The application of the serum microvesicle-asparagine endopeptidase pseudogene 1 in preparing a biomarker for diagnosing endometriosis or predicting the recurrence of endometriosis, wherein the gene sequence of the serum microvesicle-asparagine endopeptidase pseudogene 1 is shown as SEQ ID NO. 1. Also provides a kit for diagnosing endometriosis or predicting endometriosis relapse, which contains a reagent for detecting EV-LGMNP1, and comprises an upstream primer sequence, a downstream primer sequence and a probe, wherein the sequences are shown in SEQ ID NO. 5-7. It was found that EV-LGMNP1 can be detected in the serum of an endometriosis patient, the expression level of the EV-LGMNP1 is obviously higher than that of a non-endometriosis female, and the expression of the EV-LGMNP 8932 is obviously increased in a patient with endometriosis relapse, so that the serum EV-LGMNP1 can be used as a novel non-invasive biomarker for diagnosing endometriosis, predicting endometriosis relapse and monitoring disease conditions.

Description

Application of serum microvesicle-asparagine endopeptidase pseudogene 1 in diagnosis and recurrence prediction of endometriosis

Technical Field

The invention belongs to the field of medical detection, and relates to a diagnostic marker, in particular to an application of circulating microvesicle-asparagine endopeptidase pseudogene 1(EV-LGMNP1) in preparation of a marker and a diagnostic kit for diagnosing endometriosis or predicting recurrence of endometriosis.

Background

Endometriosis (EMs/EMT, abbreviated as "endometriosis") is an invasive, estrogen-dependent disease characterized by the presence of endometrial glands and stroma in other parts than the uterus. Slight endometriosis pathological changes can have tiny lesions on the surfaces of organs, and severe pathological changes can cause extensive adhesion of intestinal tracts, urinary bladders and ureters of pelvic organs. The major clinical manifestations of endometriosis are pain and infertility, to which 6% -10% of women of childbearing age are affected worldwide, seriously affecting the quality of life of the women. Meanwhile, although the internal diseases are benign diseases, the internal diseases have malignant behavior characteristics similar to tumors such as invasion and relapse, are easy to relapse, have various relapse forms, and seriously affect the physical and mental health of women of childbearing age. In addition, the pathogenesis of the internal abnormality is not completely understood so far, and the lack of specific serum markers and effective recurrence prediction means leads to the difficulty in early diagnosis and prognosis evaluation. Therefore, a biomarker for non-invasively diagnosing the internal diseases and predicting the recurrence of the internal diseases is actively explored, which is helpful for monitoring the progress of the internal diseases and promoting the early diagnosis, intervention and treatment of the internal diseases.

At present, in the biomarkers, the tissue markers have a plurality of limitations of specimen collection, incapability of continuous monitoring and follow-up visit and the like, and are not favorable for clinical development and popularization and application. The existing peripheral blood markers such as CA125 and the like have poor specificity and are not enough for early diagnosis of internal diseases. Recently, circulating biomarkers based on liquid biopsy have received attention. Three major sources of liquid biopsy are: circulating tumor DNA, circulating tumor cells, and microvesicles (exosomes). Microvesicles, which are one of the important components of the extracellular environment, are membrane-bound secretory vesicles of 30 to 150nm, and are easily separated from various biological fluids. Microvesicles are present in most body fluids of the human body such as blood, urine, ascites, etc. and can carry various bioactive substances including proteins, lipids, and genetic materials such as messenger RNA (mRNA), micro RNA (miRNA), and long non-coding RNA (lncRNA), etc. The stability of the microvesicle is extremely high, the microvesicle can be preserved for several years under various freezing, refrigerating and thawing conditions, the content is extremely rich, and each milliliter of blood plasma contains 10^8-13And a plurality of microvesicles. These advantages give the possibility of microvesicles as novel markers. It is believed that various RNAs in the circulating blood are released from the associated microvesicles, and it is due to the protection of the microvesicles that various RNAs in the circulating blood are prevented from being degraded. Therefore, the liquid biopsy technology is used for searching for specific RNA molecules from microvesicles, or can bring new opportunities for early diagnosis and disease monitoring of the internal diseases.

Of the delivery components of microvesicles, lncRNA has received much attention. The existing research shows that the microvesicle can be used as a transport carrier of functional lncRNA, induces the phenotype change in a receptor cell and participates in the generation and development of tumors; and can be used as a potential marker for tumor diagnosis and prognosis prediction. However, in the endogenous and exogenous diseases, the research report of the microvesicle lncRNA is rarely seen at home and abroad.

Disclosure of Invention

The invention aims to provide application of a serum microvesicle-asparagine endopeptidase pseudogene 1(EV-LGMNP1), and the application of the circulating serum microvesicle-asparagine endopeptidase pseudogene 1(EV-LGMNP1) aims to solve the technical problem that early diagnosis and early prediction of relapse of endometriosis are difficult in the prior art.

The invention provides an application of a serum microvesicle-asparagine endopeptidase pseudogene 1 in preparing a biomarker for diagnosing endometriosis, wherein the gene sequence of the serum microvesicle-asparagine endopeptidase pseudogene 1 is shown as SEQ ID NO. 1.

The invention also provides application of the serum microvesicle-asparagine endopeptidase pseudogene 1 in preparing a biomarker for predicting recurrence of endometriosis, wherein the gene sequence of the serum microvesicle-asparagine endopeptidase pseudogene 1 is shown as SEQ ID NO. 1.

The invention also provides a kit for diagnosing endometriosis, which contains a reagent for detecting serum microvesicle-asparagine endopeptidase pseudogene 1(EV-LGMNP1), wherein the reagent comprises an upstream primer sequence, a downstream primer sequence and a probe, the upstream primer sequence is shown as SEQ ID NO.5, the downstream primer sequence is shown as SEQ ID NO.6, and the probe sequence is shown as SEQ ID NO. 7.

The invention also provides a kit for predicting endometriosis relapse, which comprises a reagent for detecting serum microvesicle-asparagine endopeptidase pseudogene 1(EV-LGMNP1), wherein the reagent comprises an upstream primer sequence, a downstream primer sequence and a probe, the upstream primer sequence is shown as SEQ ID NO.5, the downstream primer sequence is shown as SEQ ID NO.6, and the probe sequence is shown as SEQ ID NO. 7.

Compared with the prior art, the invention has the advantages of positive and obvious technical effect. The invention discovers that: (1) the microvesicle-asparaginyl endopeptidase pseudogene 1(EV-LGMNP1) mediates the interactive dialogue of ectopic endometrium interstitial cells and macrophages, promotes the macrophages in a local heteropathy microenvironment to polarize to M2 type, and has the potential of monitoring the progress of the heteropathy. (2) On the other hand, microvesicle-asparagine endopeptidase pseudogene 1(EV-LGMNP1) is detectable in the serum of a patient with an internal disorder, and its expression level is significantly higher than that of a female who is not an internal disorder, and has the potential to diagnose an internal disorder. (3) In the patients with the recurrence of the endometriosis, the preoperative serum microvesicle-asparagine endopeptidase pseudogene 1(EV-LGMNP1) expression is obviously higher than that of the patients without the recurrence, which indicates that the index has the potential to become a biological marker for predicting the recurrence of the endometriosis preoperatively. Therefore, the liquid biopsy circulating microvesicle-asparaginyl endopeptidase pseudogene 1(EV-LGMNP1) can be a novel noninvasive biomarker for diagnosing the internal diseases and monitoring the recurrent diseases.

Drawings

FIG. 1: microvesicles (EV/Ecto-ESCs) derived from supernatants of ectopic intimal stromal cells (Ecto-ESCs) were identified.

FIG. 2 is a schematic diagram: microvesicles (EV/Ecto-ESCs) released by Ecto-ESCs are endocytosed by human myeloid leukemia monocytes (THP-1) as receptor cells, inducing THP-1 to polarize to M2 type macrophages.

FIG. 3: LGMNP1 overexpressed by Ecto-ESCs can be delivered to THP-1 of receptor cells through microvesicles, up-regulate a downstream target gene asparagine endopeptidase (LGMN), and promote THP-1 polarization to M2 type macrophages.

FIG. 4 is a schematic view of: serum EV-LGMNP1 was significantly higher in the female with endometriosis than in the female with non-endometriosis, and significantly higher in the recurrent patients than in the non-recurrent.

FIG. 5 is a schematic view of: serum EV-LGMNP1 is an independent prognostic factor for predicting the recurrence of an internal disorder.

Detailed Description

Example 1 extraction and characterization of microvesicles (EV/Ecto-ESCs) from supernatants of ectopic endometriotic stromal cells (Ecto-ESCs)

Extracting and identifying microvesicles in cell supernatant:

1) primary culture of ectopic endometrium interstitial cells (1# and 2# Ecto-ESCs)

2) Ecto-ESCs were identified using immunohistochemical methods using markers specific for vimentin (vimentin), cytokeratin (keratin), CD45, and CD 10.

3) The microvesicles were extracted with the microvesicle extraction kit ExoQuick EV precipitation solution kit (catalog No. extoc50a-1; system Biosciences, Palo Alto, California) extracts microvesicles from the cell supernatant.

4) Transmission Electron Microscope (TEM) characterization of microvesicles: and taking 20uL of the obtained micro-vesicle solution, adding 20uL PBS for dilution, then dropwise adding the solution to a sample-carrying copper net special for a microscope, standing at room temperature for 3 minutes, airing, absorbing 20uL of 3% sodium phosphotungstate solution, dropwise adding the solution to the copper net for negative dyeing for 1 minute, and absorbing excess liquid by using paper. The copper mesh was placed in an electron microscope and evacuated, and observed and photographed as shown in FIG. 1B.

5) Detecting EV/Ecto-ESCs particles by using a nanometer particle size analyzer: the extracted EV/Ecto-ESCs were diluted moderately with PBS, resuspended to 1.5-3mL, and thoroughly pipetted well. And (4) operating the machine, and measuring the particle size of the product by adopting a dynamic light scattering method under a nanometer particle size analyzer NS 300.

6) Identifying the surface marker protein of the microvesicle by Western blot: the identification is carried out by a Western blot kit by using antibodies of the micro-vesicular surface marker proteins CD9, CD63, TSG101 and HSP70 as positive markers and a Calnexin antibody as a negative control marker.

We have found that: as shown in FIG. 1A, primary Ecto-ESCs were successfully cultured. As shown in FIGS. 1B-1C, the microvesicles in the supernatant of the separated Ecto-ESCs were identified by transmission microscopy and NTA, and the microvesicles separated by a visible electron microscope were in the form of round particles with consistent size and diameter of 50-150 nm. Then Western blot is carried out for further identification, and as shown in figure 1D, the expression of the microvesicle surface marker proteins CD9, CD63, HSP70 and TSG101 can be detected, and meanwhile, the negative control Calnexin is negative. The above results indicate that the present invention successfully extracted microvesicles (EV/Ecto-ESCs) from the supernatant of Ecto-ESCs.

Example 2 microvesicles (EV/Ecto-ESCs) secreted by ectopic endometrial stromal cells are endocytosed by THP-1, a receptor cell, and induce THP-1 to polarize towards M2-type macrophages

Labeling and endocytosis experiments of microvesicles:

1) the microvesicles were labeled with the green dye PKH67(green dye, Sigma-Aldrich, st.

2) Microvesicles (EV/Ecto-ESCs) secreted by ectopic endometrial stromal cells were co-cultured with the receptor THP-1, and the endocytosis of the microvesicles was observed by a fluorescence microscope after 16 hours.

The THP-1 receptor cell is polarized to M2 type macrophage for verification:

the expression conditions of specific M1 type molecules CD86 and M2 type molecules CD206 in THP-1 cells are identified by using a real-time fluorescent quantitative PCR method and Western blot, GAPDH is used as an internal reference in qRT-PCR, and primer sequences are as follows.

TABLE 1 CD86, CD206 primer sequences

Target Gene	Primer	Sequence
			GAPDH	forward	5'-gtctcctctgacttcaacagcg-3' (shown in SEQ ID NO. 2)
	reverse	5'-accaccctgttgctgtagccaa-3' (shown in SEQ ID NO. 3)
			CD 86	forward	5'-ctgctcatctatacacggttacc-3' (shown in SEQ ID NO. 11)
	reverse	5'-ggaaacgtcgtacagttctgtg-3' (shown in SEQ ID NO. 12)
			CD 206	forward	5'-tccgggtgctgttctccta-3' (shown in SEQ ID NO. 13)
	reverse	5'-ccagtctgtttttgatggcact-3' (shown in SEQ ID NO. 14)

The reaction system is as follows:

mixing and blowing a reaction system, centrifuging at 6000rpm for a short time to remove bubbles, putting into a fluorescent quantitative PCR instrument, and performing pre-denaturation at 95 ℃ for 10 minutes; entering into circulation: denaturation at 95 ℃ for 15 seconds, annealing at 60 ℃ for 45 seconds, and extension at 75 ℃ for 15 seconds for 40 cycles; after the PCR is finished, a melting curve (60 ℃ for 1 min-95 ℃ for 15 sec-60 ℃ for 15 sec) is drawn for amplification.

We have found that: as shown in FIG. 2A, microvesicles released from Ecto-ESCs (EV/Ecto-ESCs) were endocytosed by the recipient cell THP-1, suggesting that microvesicles were transported from donor ectopic endometrial stromal cells (Ecto-ESCs) to the recipient cell THP-1. As shown in FIG. 2B, the expression of the M1-type specific molecule CD86 and the expression of the M2-type specific molecule CD206 of the receptor cell THP-1 is reduced at the mRNA and protein level after the receptor cell THP-1 internalizes the EV/Ecto-ESCs, which preliminarily suggests that the EV/Ecto-ESCs promote the THP-1 to be polarized to M2 type. (Western blot also confirmed the results of the above experiment)

Example 3 overexpression of EV-LGMNP1 secreted by donor Ecto-ESCs promotes the polarization of receptor THP-1 to M2-type macrophages and upregulates downstream target genes LGMN

Construction of over-expressed lentivirus vector and cell transfection experiment

1) Construction of a lentivirus vector: lentiviral vectors were selected and specific primers were selected according to the principle of primer design to specifically amplify the LGMNP1 gene fragment (from. gtNC-000013.11: c64959518-64957561Homo sapiens chromosome 13, GRCh38.p13 Primary Assembly, as shown in SEQ ID No. 1). Connecting the gene fragment LGMNP1 with the enzyme-cut lentiviral vector through enzyme cutting sites at two ends, transferring the gene fragment LGMNP1 into prepared competent bacteria, carrying out PCR identification on a monoclonal colony after culture, and then carrying out sequencing comparison and selecting the lentiviral vector successfully expressing LGMNP 1.

2) Lentivirus packaging and assay: the lentivirus LGMNP1 overexpression vector with correct sequencing is co-transfected into HEK293T cells by using a transgene reagent, after 12 hours of transfection, the cell is cultured for 4 hours by adding an Enhancing buffer, and then the cell is cultured for 48 hours by using a fresh culture medium, and then cell supernatant is collected, further concentrated and the virus titer is detected.

3) After Ecto-ESCs are infected, puromycin with proper concentration is added for pressure screening, then identification is carried out, total RNA is extracted from cells, and qRT-PCR is carried out to verify the transfection efficiency of LGMNP1 overexpression lentivirus in the Ecto-ESCs.

qRT-PCR confirmed that over-expressed LGMNP1 could be delivered to THP-1 via microvesicles

1) In order to ensure the stability, repeatability and reliability of the lncRNA-PCR reaction system, a probe method was adopted for detecting LGMNP1 and LGMN, GAPDH was used as an internal reference, TaqMan Universal Master Mix-II with UNG (Applied Biosystems, USA) reagents were used, and qRT-PCR detection of LGMNP1 expression was performed on eco-ESCs, EV/eco-ESCs and co-cultured THP-1 cells, with the following primers.

TABLE 2 LGMNP1, LGMN primer sequences

2) The reaction system is as follows:

2×TaqMan Universal Master Mix-II with UNG	10μL
		template cDNA	1μL(100ng)
probe-primer mix	1μL
		ddH2O	8μL
Total volume	20μL

Mixing and blowing a reaction system, and performing an amplification process: pre-denaturation at 50 ℃ for 2 min and 95 ℃ for 10 min; entering a circulation: denaturation at 95 ℃ for 15 seconds-annealing extension at 60 ℃ for 1 minute for 45 cycles; negative controls (NTC) were used to exclude false positives during the procedure.

qRT-PCR and Western blot detection of expression of CD206, CD86 and LGMN

The expression of CD86, CD206 and LGMN in THP-1 co-cultured with EVs (EV/NC vs. EV/OE) secreted by Ecto-ESCs of the control group and the overexpression group was verified by qRT-PCR and Western blot (the method was the same as the above).

We found that: as shown in fig. 3A, donor ectopic endometrial stromal cells (eco-ESCs) are able to transport LGMNP1 to recipient cell THP-1 via microvesicles; as shown in fig. 3B-3C, receptor cell THP-1 can polarize to M2-type macrophages and up-regulate down-stream target gene LGMN upon internalization of microvesicles that overexpress LGMNP 1.

Example 4 expression of endo-xenogeneic patients and controls serum microvesicles LGMNP1(EV-LGMNP1) serum microvesicle extraction procedure:

1) peripheral serum samples collected from the tissue bank were removed from-80 ℃, thawed at room temperature (15-25 ℃) and centrifuged at 3000g for 15 minutes to remove the cells or cell debris sufficiently.

2) The supernatant was aspirated with a pipette gun, transferred to a sterilized centrifuge tube, and extracted with the ExoQuick EV precipitation solution kit (catalog No. exoq5a-1; system Biosciences, Palo Alto, California) extract EVs, with reference to the instructions, in sample volumes: ExoQuick volume 4: 1 ExoQuick-EXOQ5A-1 reagent was added to the centrifuge tube.

3) The tubes were mixed by inverting and incubated at 4 ℃ for 60 minutes. Centrifuging at 1500g for 30 min, and discarding the supernatant after the centrifugation is finished. Centrifugation at 1500g was continued for 6 minutes to remove residual supernatant. Add 1 XPBS to resuspend the EVs pellet. 4) TEM, NTA and WB identification of EVs were performed as in the first section.

Expression of serum microvesicles LGMNP1(EV-LGMNP1) in patients with internal symptoms and controls

The expression level of EV-LGMNP1 is detected by using a real-time fluorescent quantitative PCR method (the specific method is the same as the method).

We found that: after the serum microvesicles were extracted, as shown in fig. 4A, serum microvesicles LGMNP1(EV-LGMNP1) of 52 patients with an abnormal condition and 21 control groups were tested by qRT-PCR method, and the results showed that the content of serum microvesicles LGMNP1(EV-LGMNP1) of the patients with an abnormal condition was significantly higher than that of the control group (P ═ 0.0106). And as shown in fig. 4B, the expression of microvesicle LGMNP1(EV-LGMNP1) was significantly increased in the relapsed population in the xenogeneic patients (P ═ 0.0004).

Example 5 correlation analysis of the expression of serum microvesicles-LGMNP 1(EV-LGMNP1) in patients with Endometapy with the clinical prognosis

Among 52 patients with allopathy, Relapse Free Survival (RFS) was analyzed using the Kaplan-Meier method in combination with one-way Log-rank and multifactor Cox regression, and 95% Confidence Intervals (CIs) were described. Based on Cox analyses conclusions, a model for recurrence prediction was constructed using the R language (Version 3.5.3) using a Nomogram (Nomogram) based on clinically significant, independently prognostic factors. In addition, according to the detection level of peripheral blood EV-LGMNP1, a Receiver operating characteristic curve (ROC) is drawn to know the value of peripheral blood EV-LGMNP expression level to recurrence prediction, a jordan index is calculated, an optimal threshold value (Cut-off value) is determined according to the maximum value, and corresponding sensitivity and specificity are calculated. P <0.05 was judged to be statistically different.

We found that: as shown in fig. 5A-5B, high expression of EV-LGMNP1 (p ═ 0.012), complete closure of the uterine pouch (p ═ 0.026) were independent prognostic factors for recurrence of endometriosis. As shown in FIGS. 5C-5E, the C-index (C-index) of the Nomogram model constructed based on this to predict the recurrence of an endometriosis was as high as 0.836 (95% confidence interval: 0.775-0.898). Internal examination of the calibration curve revealed that the model was effective in predicting RFS at 48 and 54 months post-surgery. As shown in fig. 5F, ROC results were plotted based on the expression level of EV-LGMNP 1: by taking expression difference of peripheral blood EV-LGMNP1 which is more than 2.338 times as a standard, the sensitivity for predicting the recurrence of the internal abnormality is 93.3 percent, the specificity is 75.5 percent, and the area under the curve (AUC) can reach 0.869(95 percent confidence interval: 0.774-0.963).

Sequence listing

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Claims

1. The application of the primer for detecting the serum microvesicle-asparagine endopeptidase pseudogene 1 in preparing the kit for diagnosing endometriosis comprises the gene sequence of the serum microvesicle-asparagine endopeptidase pseudogene 1 shown as SEQ ID NO.1, the kit contains a reagent for detecting the serum microvesicle-asparagine endopeptidase pseudogene 1, the reagent comprises an upstream primer sequence, a downstream primer sequence and a probe, the upstream primer sequence is shown as SEQ ID NO.5, the downstream primer sequence is shown as SEQ ID NO.6, and the probe sequence is shown as SEQ ID NO. 7.

2. The application of the primer for detecting the serum microvesicle-asparagine endopeptidase pseudogene 1 in preparing the kit for predicting the recurrence of endometriosis is disclosed, wherein the gene sequence of the serum microvesicle-asparagine endopeptidase pseudogene 1 is shown as SEQ ID No.1, the kit contains a reagent for detecting the serum microvesicle-asparagine endopeptidase pseudogene 1, the reagent comprises an upstream primer sequence, a downstream primer sequence and a probe, the upstream primer sequence is shown as SEQ ID No.5, the downstream primer sequence is shown as SEQ ID No.6, and the probe sequence is shown as SEQ ID No. 7.