CN112941149B - Multi-group chemical integration analysis-based polycystic ovarian syndrome target screening method and polycystic ovarian syndrome target - Google Patents

Abstract

The invention provides a polycystic ovarian syndrome target screening method based on multiomic integrated analysis and a polycystic ovarian syndrome target, wherein the method comprises the following steps of: (a) Performing single cell RNA sequencing and low-amount DNase sequencing on cells of a normal mouse and cells of a DHEA-induced PCOS mouse in each development stage; (b) Calculating the gene expression amount by a bioinformatics method; (c) Comparing the gene expression difference of the normal mice and the DHEA-induced PCOS mice in each development period; (d) Identifying DNA high-sensitivity sites by adopting a bioinformatics analysis method; and (e) performing correlation analysis on the gene expression information and the promoter region DNA hypersensitive sites, and screening out a gene target point with abnormal transcriptional activation in a DHEA-induced PCOS mouse. Provides an efficient and practical screening method for the polycystic ovarian syndrome target spot and provides a potential intervention target spot of the polycystic ovarian syndrome.

Description

A screening method for polycystic ovary syndrome targets based on multi-omics integration analysis and polycystic ovary syndrome targets

technical field

The invention relates to the technical field of biomedicine, in particular to a method for screening polycystic ovary syndrome targets based on multi-omics integration analysis and polycystic ovary syndrome targets.

Background technique

Polycystic ovary syndrome (PCOS) is a syndrome of endocrine disorders characterized by rare ovulation or anovulation, hyperandrogen or insulin resistance, and polycystic ovaries. It is a disease that significantly affects female fertility, metabolism, and psychology. public health issues. PCOS is very common in women of reproductive age, with an incidence rate of about 8% to 13%. Patients will have different symptoms such as menstrual disorders, hirsutism, infertility and pregnancy complications, insulin resistance, metabolic syndrome, type 2 diabetes and risk of cardiovascular disease, etc.

Clinical studies have shown that PCOS offspring are more likely to suffer from PCOS, and the risk of neonatal adverse outcomes, offspring cardiometabolic system diseases, and offspring neuropsychological developmental diseases is increased. The specific mechanism is unclear. The onset of PCOS has a genetic predisposition, and the offspring of patients have genetic susceptibility. Family genetic analysis showed that female family members with PCOS were more likely to experience menstrual disorders, ovarian enlargement, and hyperandrogenism than non-PCOS patients; while male family members were more likely to have early baldness as the phenotype.

Genome-wide association studies showed that PCOS with hyperandrogenism candidate genes (including steroid hormone synthesis genes CYP19, CYP17, CYP11a, AR, SHBG; insulin receptor substrate gene IRS; gonadotropin function and regulation related genes LH, FSH, etc. ) mutation sites, but no consensus genetic risk factors for PCOS have been found; existing studies have confirmed that PCOS patients have abnormal follicle development, abnormal epigenetic modification of lipid and steroid synthesis genes, and lack of genome-wide analysis of offspring embryos before and after implantation ; Observational studies on PCOS offspring are increasing. PCOS offspring show metabolic abnormalities in childhood. Considering the bias and heterogeneity of case data analysis, the basic research mechanism of disease transmission is still unclear, and the target of early intervention is not clear .

In view of this, the present invention is proposed.

Contents of the invention

The purpose of the present invention is to provide a polycystic ovary syndrome target screening method and polycystic ovary syndrome target based on multi-omics integration analysis, and to provide an efficient and practical polycystic ovary syndrome target screening method , and provides a potential intervention target for polycystic ovary syndrome.

The technical scheme provided by the invention is as follows:

A method for screening polycystic ovary syndrome targets based on multi-omics integrated analysis, the method comprising:

(a) Single-cell RNA sequencing and low-quantity DNase sequencing were performed on the cells of normal mice and DHEA-induced PCOS mice at each developmental stage;

(b) Calculating the amount of gene expression by means of bioinformatics;

(c) compare the gene expression differences of normal mice and DHEA-induced PCOS mice at each developmental stage;

(d) Using bioinformatics analysis methods to identify DNA hypersensitive sites; and

(e) Association analysis of gene expression information and DNA hypersensitivity sites in the promoter region was carried out to screen out the gene targets with abnormal transcriptional activation induced by DHEA in PCOS mice.

In one embodiment, in step (a), the cells at each developmental stage include: germinal vesicle (GV) stage oocyte, meiosis second metaphase (MII) oocyte, 2-cell embryo, 4-cell Embryos, 8-cell embryos, morula cells, and blastocyst cells;

Preferably, the sampling of cells at each developmental stage is carried out from a plurality of mouse samples;

More preferably, the total number of cells at each developmental stage is >20.

In one embodiment, after the step (c), the method further includes the step of screening genes that DHEA induces early activation of PCOS mice at the 2-cell embryo stage;

Preferably, the screening threshold is expressed in at least 80% of DHEA-induced PCOS mice (ie FPKM>1); and expressed in at most 20% of normal mouse samples (ie FPKM>1).

DHEA-induced early activation of PCOS mice (also known as DHEA-treated mice) at the 2-cell embryonic stage refers to genes that are not expressed in normal mice but expressed in DHEA-treated mice.

In one embodiment, in step (a), Illumina HiSeq 4000 is used for sequence determination after library construction to obtain single-cell RNA-seq data (scRNA-seq) and low-volume DNase-seq data (liDNase-seq).

In one embodiment, in step (b), the scRNA-seq data is quality controlled using Trim Galore (v 0.6.5) software to remove low-quality sequencing reads. Use STAR (v 2.7.3a) software to perform genome alignment on the quality-controlled scRNA-seq data, and obtain the genome alignment result bam file. The expression information of each gene was calculated using Cufflinks (v 2.2.1) software.

In one embodiment, in step (c), the gene that defines Cufflinks calculation gene expression quantity result FPKM>1 is expressed gene;

The number of expressed genes in each developmental period was calculated respectively, and the number of expressed genes in each developmental period of normal mice and DHEA-induced PCOS mice was compared.

By analyzing the number of expressed genes at each developmental stage and comparing them with normal mice, we found the period when the transcriptional activity in DHEA-treated mouse cells was significantly increased. Subsequent further screening of genes activated in advance in DHEA-treated mice at this cell stage.

In one embodiment, the step (d) comprises using Trim Galore software to perform quality control on low-volume DNase-seq data to remove low-quality sequencing reads;

Use STAR software to perform genome comparison on low-volume DNase-seq data after quality control;

Use Samtools software to perform quality control on the alignment result files, and remove sequencing reads whose alignment quality is lower than 20;

Duplicate sequencing reads generated by polymerase chain reaction were removed using PICARD software;

DNA hypersensitive sites were identified using the hotspot2 algorithm.

In a specific embodiment, the step (d) includes using a local script file to calculate the liDNase-seq signal intensity (liDNase-seq density) of the gene promoter region, that is, the number of reads measured in every million bases .

In one embodiment, said step (e) comprises using the t hypothesis test to analyze the enrichment of promoter opening genes in early transcription activation genes in 2-cell embryo stage DHEA-induced PCOS mice;

Among them, the promoter-opening gene is a gene whose promoter region contains DNA hypersensitive sites (genes are divided into promoter-opening and promoter-not-opening genes according to whether the gene promoter region contains DHS).

The early transcription genes of 2-cell embryonic stage in DHEA-treated mice were screened: the promoters were opened in GV phase cells of DHEA-treated mice but not in normal mouse GV phase cells.

In one embodiment, before step (a), the present invention also includes the step of cultivating normal mice and establishing a dehydroepiandrosterone-induced PCOS mouse model (DHEA mice for short), specifically selecting a 3-week-old Female C57BL/6J mice were injected with dehydroepiandrosterone (6mg/100g) daily in the neck for 20 days, and the model was successfully established if there was no obvious estrous cycle in the vaginal smear.

The present invention also provides a polycystic ovary syndrome target, the target is Oct4 gene or its expression product Oct4 protein.

The present invention also provides the use of the Oct4 gene or its expression product Oct4 protein as a molecular target or drug action target in screening and preparing polycystic ovary syndrome medicines.

According to the above uses of the present invention, those skilled in the art can also use the reagents for inhibiting or silencing the expression of Oct4 gene in the preparation of polycystic ovary syndrome medicines.

Beneficial effect:

The present invention provides a brand-new polycystic ovary syndrome target screening method based on multi-omics integrated analysis. The method combines single-cell RNA sequencing and low-quantity DNase sequencing to build a library, and analyzes genes through bioinformatics methods. The expression level and expression difference, and the association analysis between the differentially expressed genes and the promoter region DHS, effectively screened out the early transcription activation genes in DHEA-treated mice at the 2-cell embryonic stage.

The invention provides a potential novel target Oct4 gene or its expression product for treating polycystic ovary syndrome, and has a strong possibility of clinical application.

Description of drawings

In order to more clearly illustrate the specific implementation of the present invention or the technical solutions in the prior art, the following will briefly introduce the accompanying drawings that need to be used in the specific implementation or description of the prior art. Obviously, the accompanying drawings in the following description The drawings show some implementations of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative work.

Fig. 1 is the schematic diagram of the PCOS model modeling process that the embodiment of the present invention provides;

Fig. 2 is the extraction schematic diagram of the GV stage and MII stage oocyte provided by the embodiment of the present invention;

Fig. 3 is the schematic flow chart of DNase library construction that the embodiment of the present invention provides;

Figure 4 is a diagram showing the difference in gene expression between normal mice and DHEA-treated (DHEA-treated) mice at each developmental stage provided by the embodiments of the present invention;

Figure 5 shows that after Oct4 gene interference, the cell development stagnates at the 4-cell stage, while the control group can develop into blastocysts (where siCTR indicates that the invalid interference sequence was injected at the 2-cell stage of the control group, and siOct4 indicates that Oct4 was knocked out in the DHEA-treated 2-cell embryos ).

detailed description

The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, but not all of them. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

Example 1: Screening method for polycystic ovary syndrome intervention targets based on multi-omics integration analysis

1.1 PCOS model modeling

Normal mice were cultivated to establish a dehydroepiandrosterone (DHEA)-induced PCOS-like mouse model (DHEA mouse for short). Specifically:

Eight 3-week-old female C57BL/6J mice were selected and divided into two groups, a normal control group and a dehydroepiandrosterone (DHEA) group, with 4 mice in each group. Female C57BL/6J mice were obtained through conventional purchase channels (purchased from Vital River Experimental Animal Technology Co., Ltd. (Beijing, China)). All animal experiments complied with the rules and guidelines of the local Animal Ethics Committee and the Animal Care and Use Committee of Peking Union Medical College Hospital. The study protocol was approved by the Animal Care and Use Committee of Peking Union Medical College Hospital.

Adaptive culture until the 25th day (recorded as D25), the mice in the normal control group were injected with 0.1ml mixed sesame oil in the neck, and the administration continued for 20 days; the DHEA group was injected with DHEA solution (DHEA 6mg/100g+0.1ml mixed sesame oil) in the neck , Continuous administration for 20 days. Body weight changes were recorded daily. Daily vaginal smears were performed from D38 to D45 to determine the estrous cycle. Glucose tolerance test was performed on D44.

After administration, mice in the normal control group, according to the results of vaginal smears, collected serum after fasting during the estrus period, killed the mice by dislocation, and kept the ovaries, abdominal fat, and liver of the mice for further examination. If the mice were successfully modeled, there would be no obvious estrous cycle. Serum was collected after fasting on D45, and the mice were killed by dislocation, and the ovaries, abdominal fat, and liver of the mice were kept for further examination.

The criteria for the successful establishment of the polycystic ovary syndrome model: typical polycystic lesions in the ovary of female mice, elevated androgen and other morphological and endocrine changes. The schematic diagram of the PCOS model modeling process is shown in Figure 1.

1.2 Collection of cells at each developmental stage

Collect germinal vesicle (GV) oocytes, meiotic metaphase (MII) oocytes, 2-cell embryos, 4-cell embryos, 8-cell embryos, morula cells, and cysts from normal mice and DHEA mice Embryonic cells, cells in each developmental stage were taken from multiple mouse samples, and the total number of cells in each developmental stage was >20.

Wherein the extraction method of GV stage and MII stage oocyte (see Fig. 2) is as follows: get each 3 mice of normal control group and DHEA group, use pregnant horse serum gonadotropin (Pregnant Mare Serum Gonadotropin, PMSG) 10IU at D45 After 48 hours of ovulation induction, the mice were sacrificed by dislocation, and both ovaries were taken out by laparotomy, washed three times with the prepared M199, and then punctured the ovarian tissue with a 1 ml sterile insulin syringe (30g needles) under a dissecting microscope , expose the oocytes in the GV stage, suck the oocytes in the GV stage with a Pasteur pipette, put them into the hyaluronidase liquid, and repeatedly blow and beat them for 30 seconds with a thin Pasteur pipette to remove the oocytes The surrounding granule cells were quickly transferred into M199, repeatedly pipetted with a thinner Pasteur pipette to remove the remaining granule cells, and finally transferred to PBS and washed once, then transferred to EP tubes.

Three mice in the normal control group and three mice in the DHEA group were injected with 10 IU human chorionic gonadotropin (HCG) 48 hours after ovulation induction with PMSG 10 IU/mouse on D45, and the mice were killed by dislocation at 18 hours. Take out the oviducts on both sides by laparotomy, wash them three times repeatedly with the prepared M199, and then puncture the oviducts with a 1 ml sterile syringe under a dissecting microscope to expose the oocytes in the MII stage, and absorb the oocytes in the MII stage with a Pasteur pipette Put the cells into the hyaluronidase liquid, blow and beat repeatedly with a thinner Pasteur pipette for 30 seconds to remove the granulosa cells around the oocyte, quickly move them into M199, and repeatedly blow and beat with a thinner Pasteur pipette, Remove the remaining granule cells, transfer to PBS and wash once, then transfer to EP tube.

1.3 Single-cell RNA sequencing and low-volume DNase sequencing library construction for cells at various developmental stages

Illumina HiSeq 4000 was used for sequence determination to obtain single-cell RNA-seq data (scRNA-seq) and low-volume DNase-seq data (liDNase-seq).

Single-cell transcriptome scRNA-seq library preparation includes:

1) Cell sample preparation: Use a Pasteur pipette to absorb germ cells at different stages, resuspend them in 1×PBS, and place them at the bottom of the EP tube. Operate on ice.

2) Synthesis of first-strand cDNA: prepare Lysis buffer, add it to the EP tube containing a single cell, add Oligo(dT)VN primer, dNTP Mix, mix gently and react in a PCR instrument. Reaction conditions: extension at 72°C for 3 minutes; immediately place on ice for 2 minutes. Prepare the reverse transcription reaction system, carry out 90min at 42°C; 15min at 70°C; cool at 4°C.

3) Amplification and purification of full-length cDNA: Prepare a PCR amplification system, mix well and react in a PCR instrument. Reaction conditions: denaturation at 98°C for 10s; annealing at 65°C for 15s; extension at 72°C for 6min (16 cycles); extension at 72°C for 5min; cooling at 4°C.

After purification by magnetic bead separation, Agilent 2100 Bioanalyzer was used for product quality control and identification.

4) Library construction and loading: After the above-mentioned product fragments were operated according to the DNA library preparation kit, they were loaded onto the Illumina Hiseq platform, and sequenced in PE150 mode.

The DNase library construction process is shown in the literature reports of Lu et al., 2016; Gao et al., 2018; Jin et al., 2015. The specific flow diagram is shown in Figure 3.

1.4. Gene expression was calculated using bioinformatics analysis methods.

The specific process includes: using Trim Galore (v 0.6.5) software to perform quality control on scRNA-seq data and remove low-quality sequencing reads. Use STAR (v 2.7.3a) software to perform genome alignment on the quality-controlled scRNA-seq data, and obtain the genome alignment result bam file. The expression information of each gene was calculated using Cufflinks (v 2.2.1) software.

1.5. Comparison of gene expression differences between normal mice and DHEA-treated (DHEA-treated) mice at each developmental stage.

Define Cufflinks to calculate gene expression results of FPKM>1 genes as expressed genes, respectively calculate the number of expressed genes in each developmental period, and compare the number of expressed genes in each developmental period of normal mice and DHEA-treated mice (as shown in Figure 4 ). It was found that in 2-cell embryos, the number of genes expressed in DHEA-treated mice was higher than that of normal mice, indicating that the transcriptional activity in DHEA-treated mouse cells was significantly increased at the 2-cell stage.

1.6 Screen the genes activated in advance in DHEA-treated mice at the 2-cell stage, that is, genes that are not expressed in normal mice but expressed in DHEA-treated mice.

The specific screening thresholds are:

1. Expressed in at least 80% of DHEA-treated mouse samples (ie FPKM>1);

2. Expressed in at most 20% of normal mouse samples (ie FPKM>1).

A total of 1594 such genes were obtained.

1.7 Use bioinformatics analysis methods to identify DNA hypersensitive sites (DHS).

The specific process includes: using Trim Galore (v 0.6.5) software to perform quality control on liDNase-seq data and remove low-quality sequencing reads. Genome alignment was performed on the quality-controlled liDNase-seq data using STAR (v 2.7.3a) software. Use Samtools software to perform quality control on the alignment result files, and remove sequencing reads with alignment quality lower than 20. Duplicate sequencing reads generated by polymerase chain reaction (PCR) were removed using PICARD software. DNA hypersensitive sites (DHS) were identified using the hotspot2 algorithm. Use the local script file to calculate the liDNase-seq signal intensity (liDNase-seq density) of the gene promoter region, that is, the number of reads measured per million bases.

1.8 Association analysis of gene expression and promoter region DHS.

According to whether the gene promoter region contains DHS, the genes are divided into promoter open and promoter closed genes. The enrichment of promoter open genes among early transcriptionally activated genes in DHEA-treated mice at 2-cell stage was analyzed using t-hypothesis test. Promoters were defined as ±2 kb around the TSS of annotated genes (GENCODE vM9, Ensembl 84). DHSs overlapping the promoter by at least 1 bp were identified as promoter DHSs.

It was found that 433 early transcribed genes of 2-cell DHEA-treated mice (see Table 1 below) had their promoters open in GV phase cells of DHEA-treated mice but not in normal mouse GV phase cells. It shows that in DHEA-treated mice, the abnormal opening of the GV promoter will affect the transcriptional activation at the 2-cell stage.

Table 1. 433 pre-transcribed genes in 2-cell DHEA-treated mice:

Embodiment 2: polycystic ovary syndrome target: human Oct4 gene or its expression product Oct4 protein

In DHEA-treated 2-cell embryos, the transcription factor Oct4 was overexpressed from 2- to 8-cell embryo stages. Oct4 expression was more than 200-fold higher in DHEA-treated 2-cell embryos than in control 2-cell embryos. A clear increase in DHS signal was observed in the DHEA-treated GV stage 12-kb downstream of the Oct4 promoter.

Taking the Oct4 gene (one of the 433 genes in 1.8 of Example 1) as an example to verify the phenomenon of transcriptional activation at the 2-cell stage, a small RNA interference experiment (Small interfering RNA) was carried out. siRNA was injected into 1-cell embryos, and siRNA reagents for Oct4 were purchased from Thermo (Thermo, s71991, 71993); negative control siRNA (Thermo, s4390843) was used. Approximately 10 pl of siRNA was injected per embryo. Injected embryos with normal morphology for scRNA-seq were harvested at the late 2-cell stage. Three groups of siRNA-treated embryos were pooled for one scRNA-seq library preparation assay. Two biological replicates were used.

Single-cell RNA sequencing was performed on 2-cell embryos before and after the interference experiment to confirm that the Oct4 gene was silenced. Cell experiments verified that after Oct4 gene interference, cell development stagnated at the 4-cell stage, while the control group could develop into blastocysts, as shown in Figure 5.

The biological pathways affected by Oct4 overexpression in DHEA-treated mice were statistically analyzed by biological pathway enrichment (GO). It was found that abnormally expressed genes were significantly enriched in biological processes such as lipid metabolism and steroid synthesis, which are the phenotypic characteristics of PCOS, indicating that Oct4 is a potential intervention target for PCOS.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present invention, rather than limiting them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: It is still possible to modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the technical solutions of the various embodiments of the present invention. scope.

Claims (3)

1. A screening method of polycystic ovary syndrome targets based on multigroup chemical integration analysis, which comprises the following steps:

(a) Performing single-cell RNA sequencing and low-amount DNase sequencing on cells of a normal mouse and cells of a DHEA-induced PCOS mouse in each development period;

(b) Calculating the gene expression level by a bioinformatics method;

(c) Comparing the gene expression difference of the normal mice and the DHEA-induced PCOS mice in each development period;

(d) Identifying DNA high-sensitivity sites by adopting a bioinformatics analysis method; and

(e) Performing correlation analysis on the gene expression information and the promoter region DNA hypersensitive sites, and screening out a gene target point with abnormal transcriptional activation in a DHEA-induced PCOS mouse;

in step (a), each of the developmental stage cells comprises: oocytes in a germinal vesicle stage, oocytes in a meiotic second metaphase, 2-cell embryos, 4-cell embryos, 8-cell embryos, morula cells and blastocyst cells;

in step (b), using Trim Galore software to perform quality control on RNA-seq data and removing low-quality sequencing reads; performing genome comparison on the RNA-seq data after quality control by using STAR software to obtain a genome comparison result; calculating the expression amount information of each gene by using Cufflinks software;

in the step (c), defining the gene with Cufflinks gene expression quantity result FPKM >1 as an expression gene; respectively calculating the number of the expression genes in each development period, and comparing the number of the expression genes in each development period of the normal mice and the PCOS mice induced by DHEA;

the method further comprises the step of screening for a gene that induces premature PCOS mice activation by DHEA in 2-cell embryonic stage after step (c); the threshold for said screening is at least 80% DHEA-induced PCOS mice expression; and up to 20% of normal mouse samples;

the step (d) comprises performing quality control on the low-level DNase-seq data by using Trim Galore software, and removing low-quality sequencing reads; performing genome comparison on the low-amount DNase-seq data after quality control by using STAR software; using Samtools software to compare and control the quality of the result file, and removing sequencing reads with the comparison quality lower than 20; removing repeated sequencing reads generated by polymerase chain reaction using PICARD software; identifying DNA hypersensitive sites by using a hotspot2 algorithm;

said step (e) comprises analyzing the enrichment of promoter opening genes in premature transcriptional activator genes in DHEA-induced PCOS mice in 2-cell embryonic phase using a t-hypothesis test; wherein the promoter open gene is a gene of which the gene promoter region contains a DNA hypersensitive site.

2. The method of claim 1, wherein in step (a), the each developmental stage cell is sampled from a plurality of mouse samples; the total number of cells in each developmental stage was >20.

3. The method of claim 1, wherein in step (a), the single cell RNA-seq data and the low level DNase-seq data are obtained by sequencing using Illumina HiSeq 4000 after library construction.

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