CN117599184A - Application of ZNF98 gene expression in knockdown cells in preparation of medicines for treating vascular malformation - Google Patents

Abstract

The invention relates to the technical field of medical products, in particular to application of ZNF98 gene expression in knockdown cells in preparation of a medicament for treating vascular malformation. The scheme of the invention comprises the application of knocking down the expression of the ZNF98 gene in cells in preparing medicaments for preventing and/or treating vascular malformations, a kit for preventing and/or treating vascular malformations and nano medicaments for preventing and/or treating vascular malformations. The nano-drug ZNF98-siRNA@CDM-PEG-PDPA carrying ZNF98 prepared by the invention realizes the effect of specifically inhibiting or knocking out the ZNF98 gene, and the research is expected to provide theoretical basis and experimental basis for diagnosis and treatment of VaMs, and can be used as a drug for treating and preventing the VaMs.

Description

Application of ZNF98 gene expression in knockdown cells in preparation of medicines for treating vascular malformation

Technical Field

The invention relates to the technical field of medical products, in particular to application of ZNF98 gene expression in knockdown cells in preparation of a medicament for treating vascular malformation.

Background

Vascular malformations (Vascular malformations, vaMs) are complex vascular abnormalities characterized by neovascular and vascular sprouting/division imbalance (normally, induction and inhibition of angiogenesis/sprouting are in dynamic balance), and vascular network disorders, the etiology of which is not completely defined. Recent statistical studies have shown that VaMs have a global annual incidence of about 1% to 1.5%. Vascular diseases can be classified into hemangiomas and vascular malformations based on classification methods of vascular endothelial cell biological properties according to the typing criteria established by the international society of vascular disease research (International Society for the Study of Vascular Anomalies, ISSVA) revision guidelines in 2018. Vascular deformity is classified into capillary deformity, lymphatic deformity, venous deformity, arteriovenous fistula and complex vascular deformity according to the pathophysiological mechanism and the difference of anatomical parts. VaMs often exist at birth in patients, and in clinical symptoms, vaMs patients show chronic pain, skin/organ bleeding, and serious cases can develop cerebral apoplexy and multiple organ failure, etc. In addition, vaMs can occur on the body surface or in deep tissues and organs, and can have hidden disease and progress for visceral vascular lesions, and the lesion degree is complex and is more difficult to diagnose early. At present, the clinical treatment means aiming at vascular diseases comprise surgical excision, isotope treatment, laser treatment, interventional embolism, hardening treatment and the like, which belong to symptomatic treatment and can not radically treat the etiology of the diseases. Diagnosis and treatment of this type of disease presents a significant challenge to the clinician due to the limited therapeutic strategies for VaMs. Therefore, the research of the etiology of the VaMs, the research of key pathogenic genes and the elucidation of specific mechanisms thereof, and the establishment of high-sensitivity early detection and targeted treatment means are key to improving the prognosis of VaMs patients, and are one of hot spots of research in the field of vascular diseases.

More and more studies have shown that VaMs are closely related to genetic mutations, the genetic variation of which is an important causative factor of VaMs. For example: human TIE2, PIK3CA gene mutations are associated with venous malformations; ENG gene mutation is associated with hereditary arteriovenous malformations; the FLT1 gene mutation is associated with lymphatic malformation. Furthermore, our previous studies have shown that mutation of the DDX24 gene results in the occurrence of VaMs in a multiple organ venous and lymphatic deficiency syndrome (Multi-organ venous and lymphatic defect syndrome, MOVLD). The VaMs are mostly hereditary, and vascular abnormalities can be caused by germ cell or/and somatic mutation, and are regulated by various biological function related signal pathways, including VEGFA/VEGFR2, PI3K/AKT/mTOR pathway, RAS/RAF/MEK/ERK pathway, HGF/c-Met pathway and the like. Among them, the second hit theory in genetics, which further explains the later age of onset of some VaMs patients, suggests that these patients need to develop germ cell mutation-somatic mutation in succession in both alleles of the same cell to develop vascular structure/dysfunction. In summary, further analysis of genetic variation of VaMs can elucidate its disease-associated genetic structure, information transfer, gene expression and regulation, and its effects on cellular biological function, from etiology and DNA/chromosome levels. In addition, although there are pathogenic genes such as TIE2/ENG in VaMs, there are still a significant part of VaMs patients without known genetic mutation, and most of the genetic locus mutations reported at present are common mutations, but the role of rare mutation sites of related genes in VaMs is still unclear.

The protein coded by the ZNF98 gene is an important member of a Zinc finger structural domain protein family (Zinc-finger protein family), and can selectively bind to a specific target structural domain due to the structural characteristics of the protein family, so that the protein family member plays an important role in the biological processes of gene transcription regulation, signal transduction, ubiquitination mediated protein degradation, DNA damage repair, actin targeting regulation, cell migration function and the like. According to published literature reports, the protein coded by ZNF24 can inhibit the expression of vascular endothelial growth factor (Vascular endothelial cell growth factor, VEGF), cause vascular function defects and then inhibit the neogenesis of tumor blood supply blood vessels; reduced expression of the zinc finger structural protein BAZF can impair vascular neo-sprouting and capillary remodeling functions. The above studies suggest that ZNF98 plays an important role in the initiation of VaMs mediated by vascular endothelial cell dysfunction. However, the precise mechanism of action of ZNF98 in the development of VaMs is not known, so that there is a lack of research and diagnosis means for diseases possibly related to ZNF98, and thus it is necessary to study the relationship between VaMs and ZNF98, and the application of ZNF98 to early detection, targeted therapy and prognosis of VaMs.

The nano-drug has more advantages in mediating the targeted transportation of short nucleic acid sequences, antisense nucleic acid sequences and small molecular substances, and has the characteristics of self-assembly, high stability, high loading efficiency, high biocompatibility and the like. No nano-drug was found in the treatment of vascular malformations.

Disclosure of Invention

The invention aims at defining the precise action mechanism of ZNF98 in VaMs, and by researching the relationship between the VaMs and the ZNF98, the invention defines that the non-synonymous mutation proportion of the ZNF98 gene is lower and the score of the function loss intolerance rate of the coded protein is higher. ZNF98 plays an important role in vascular endothelial cell migration and tube formation, and the small interfering RNA, dsRNA, microRNA and antisense nucleic acid knockdown ZNF98 can cause the function of the VEGF signal path in terms of corresponding change of genes, so that the migration and tube formation capacity of umbilical vein endothelial cells (HUVEC) and hepatic sinus gland endothelial cells (HHSEC) is obviously weakened, and the function of the zebra fish tail venous plexus in generating new blood vessel change is important in researching the onset and treatment of VaMs. Meanwhile, a nano drug ZNF98-siRNA@CDM-PEG-PDPA for treating and preventing vascular malformation is prepared, and the effect of specifically inhibiting or knocking out ZNF98 gene is realized, so that a new method is provided for early detection, targeted treatment and prognosis of VaMs. In particular to the application of knocking down the expression of ZNF98 gene in cells in preparing medicaments for treating vascular malformations.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides an application of knocking down ZNF98 gene expression in cells in preparing medicaments for preventing and/or treating vascular malformations.

Preferably, the cells are endothelial cells;

the endothelial cells are umbilical vein endothelial cells or hepatic sinus gland endothelial cells.

Preferably, knocking down the expression of ZNF98 gene in liver sinusoidal gland endothelial cells causes a change in the gene in VEGF signaling pathway.

Preferably, the substance that knocks down ZNF98 gene expression in the cell is a small interfering RNA, dsRNA, microrna or antisense nucleic acid.

The invention also provides application of knocking down the expression of the ZNF98 gene in cells in preparing medicaments for inhibiting the expression of the ZNF98 or VEGF channel related genes.

The invention also provides a kit for preventing and/or treating vascular malformations, which comprises a substance for knocking down ZNF98 gene expression in cells.

Preferably, the substance knockdown of ZNF98 gene expression in the cell is a small interfering RNA, dsRNA, microrna or antisense nucleic acid.

The invention also provides a nano-drug for preventing and/or treating vascular malformations, which comprises a substance for knocking down ZNF98 gene expression in cells and an empty nano-carrier;

the substances for knocking down ZNF98 gene expression in the cells are small interfering RNA, dsRNA, micro RNA or antisense nucleic acid;

the empty nano-carrier is CDM-PEG-PDPA.

The invention also provides a preparation method of the nano-drug for preventing and/or treating vascular malformations, which comprises the following steps:

(1) Mixing a substance for knocking down ZNF98 gene expression in cells with water to obtain a target substance;

(2) Mixing the empty nano carrier with acetone to obtain a mixture, and mixing and stirring the mixture with water for 3-5 hours to obtain an intermediate carrier;

(3) And mixing the targeting substance with the intermediate carrier, and extruding to obtain the nano-drug. Preferably, the rotational speed of the mixing and stirring in the step (2) is 500-700 rpm; the number of times of extrusion in the step (3) is 18-22.

The invention provides an application of knocking down ZNF98 gene expression in cells in preparing medicaments for treating vascular malformations. The scheme of the invention has the following advantages:

according to the invention, through deep sequencing of the blood sample of a VaMs patient, the ZNF98 gene is subjected to rare mutation and has related mutation sites, and the change of the expression level of ZNF98 in human umbilical vein endothelial cells and human hepatic sinus gland endothelial cells is closely related to the growth and development of blood vessels. ZNF98 can mediate vascular endothelial cell migration and altered tubular function through downstream VEGF pathways leading to the occurrence of VaMs. The effect of specifically inhibiting or knocking out ZNF98 genes is realized by preparing a nano drug ZNF98-siRNA@CDM-PEG-PDPA carrying ZNF98, and the research is expected to provide theoretical basis and experimental basis for diagnosis and treatment of VaMs, and can be used as a drug for treating and preventing the VaMs.

Drawings

FIG. 1 is a synthetic route diagram of Allys-PEG-Br.

FIG. 2 is a synthetic route diagram of HO-PEG-Br.

FIG. 3 is a synthetic route diagram for HO-PEG-PDPA.

FIG. 4 is a CDM-PEG-PDPA synthetic route pattern.

FIG. 5 is a flow chart of exon depth sequencing, data quality control and candidate disease-associated gene screening analysis.

FIG. 6 shows the quality control screening of sequencing results using Principal Component Analysis (PCA) and affinity correlation analysis (plink) (A shows the screening of poor quality gene mutation sequence data using the PCA method, and B shows the screening of poor quality gene mutation sequence data using the plink method).

FIG. 7 is a screen for mutant genes in full exon depth sequencing of VaMs blood samples using rare mutation enrichment analysis.

FIG. 8 is a schematic diagram of the linkage/interaction of member-encoded proteins in Cluster1 gene Cluster using STRING (A represents the construction of a protein interaction network using STRING and 704 genes with rare mutation sites, B represents the loss-of-function intolerance score of Cluster1 gene Cluster (in red box), C represents the linkage/interaction of member-encoded proteins in Cluster1 gene Cluster, and D represents the non-synonymous mutation ratio of Cluster1 gene Cluster).

FIG. 9 shows the effect of ZNF98 expression on cell migration and tube formation in knockdown Human Umbilical Vein Endothelial Cells (HUVEC) and human liver sinusoidal gland endothelial cells (HHSC) (A is the expression level of ZNF98 in HUVEC cells transfected with siRNA or control siRNA (siNC), B is the expression level of ZNF98 in HHSC cells transfected with siRNA or control siRNA (siNC), C shows the effect of knockdown ZNF98 on HUVEC cell viability, D shows the effect of knockdown ZNF98 on HHSC cell viability, E shows the effect of knockdown ZNF98 on HUVEC and HHSC cell migration, F shows the effect of HUVEC and HHSC cell migration number statistical analysis results after knockdown ZNF98, G-H shows the effect of HUVEC cell tube formation after knockdown ZNF98, and I-J shows the effect of HHSC cell tube formation after knockdown ZNF 98).

FIG. 10 shows the effect of knockdown ZNF98 gene expression on neovascular development in the zebra fish tail venous plexus (A indicates RT-PCR products after knockdown ZNF98 gene expression, B indicates the growth and cardiovascular formation of the zebra fish tail venous plexus after knockdown ZNF98 gene, C indicates the size statistics of the zebra fish tail venous plexus region).

FIG. 11 shows the effect of ZNF98 knockdown on VEGF signaling pathway (A shows the heat map of the differential expression gene after ZNF98 knockdown, B shows the volcanic map of the differential expression gene after ZNF98 knockdown, C shows the result of enrichment of VEGF signaling pathway in GO and KEGG pathways after ZNF98 knockdown, and D shows the change of qPCR detection related candidate genes after ZNF98 knockdown).

Fig. 12 shows the shape and particle size of the nano-drug (a represents the shape of the nano-drug and B represents the particle size of the nano-drug).

FIG. 13 is a graph showing the effect of the nanomaterials on C57BL/6J vascular malformed mice (A is a graph showing the Digital Subtraction Angiography (DSA) of the portal vein puncture line in the free hepatic portal area of the mice), the red arrow is the liver of the mice, the green arrow is the hepatic portal segment of the portal vein of the mice, B is a graph showing the Tie2-wt control group of the portal vein DSA, the red arrow points to the region where normal portal vein and branches thereof are visible, C is a graph showing the Tie2-L914F experimental group of the VaMs mouse portal vein DSA, the red arrow points to the region where abnormal/malformed portal vein and branches thereof are visible, D is a graph showing the region where abnormal portal vein and branches thereof are significantly reduced, and most of the VaMs mouse portal vein and branches thereof are normal portal vein and branches thereof) of the experimental group of the nanomedics treated.

Detailed Description

In the invention, the preparation method of the empty nano carrier CDM-PEG-PDPA comprises the following steps:

(1) Preparation of alpha-double bond-epsilon-bromo polyethylene glycol (Allys-PEG-Br)

Placing a proper amount of alpha-double bond-epsilon-hydroxyl polyethylene glycol and 2-bromoisobutyl acyl bromide into chloroform, and stirring for 24 hours by a magnetic rotor after the alpha-double bond-epsilon-hydroxyl polyethylene glycol and 2-bromoisobutyl acyl bromide are fully dissolved; the solution obtained is then precipitated twice with cold diethyl ether and washed; and filtering to obtain a solid product, and drying the solid product under vacuum to obtain a final product, wherein the appearance of the final product is white powder. The synthetic route of Allys-PEG-Br is shown in FIG. 1.

(2) Preparation of alpha-hydroxy-epsilon-bromopolyethylene glycol (HO-PEG-Br)

2-mercaptoethanol having a concentration of 20mmol and a volume of 2.17ml and a-double bond-. Epsilon. -bromopolyethylene glycol having a concentration of 2mmol, 7.65g by mass, were sufficiently dissolved in 1, 4-dioxane, AIBN (2, 2' -bis (2-methylpropanenitrile)) having a concentration of 2mmol and a mass of 0.49g by mass was added as a reaction inducer, and reacted sufficiently at a temperature of 70℃for 48 hours. The obtained reaction solution was taken and chloroform was added thereto to obtain a reaction solution. The reaction solution was washed well with saturated ammonium chloride solution, and then the chloroform organic phase solution was concentrated and precipitated with cold diethyl ether. The final product was dried under sufficient vacuum and the final product was pale yellow powder in appearance. The synthetic route for HO-PEG-Br is shown in FIG. 2.

(3) Preparation of hydroxy-polyethylene glycol-poly (2-isopropylamino) ethyl methacrylate (HO-PEG-PDPA)

Alpha-double bond-epsilon-bromine polyethylene glycol with the concentration of 0.05mmol and the mass of 0.13g, cuprous bromide with the concentration of 0.10mmol and the mass of 14.3mg, pentamethyldiethylenetriamine with the concentration of 0.10mmol and the volume of 22 mu L, 2-isopropyl amino ethyl methacrylate with the concentration of 2.5mmol and the volume of 592 mu L, azodiisobutyronitrile with the concentration of 0.01mmol and the mass of 1.6mg are repeatedly frozen and melted for three times in dioxane, and the reactant is placed in a reaction tube at 60 ℃ for sealing reaction for 24 hours. The reaction tube is placed at low temperature to quickly cool down. Then, the product was placed in a dialysis bag at room temperature, the dialysis bag (molecular weight 3.5 kDa) was placed in a large amount of deionized water, and after sufficient dialysis (about 48 hours), the sample was freeze-dried, and the final product was in the form of a white powder. The synthetic route for HO-PEG-PDPA is shown in FIG. 3.

(4) Synthesis of 2-propionic acid-3-methylmaleic anhydride-polyethylene glycol-poly (2-isopropylamino) ethyl methacrylate (CDM-PEG-PDPA)

And (3) acyl chloride 2-propionic acid-3-methyl maleic anhydride, and fully reacting the product with hydroxy-polyethylene glycol-poly (2-isopropylamino) ethyl methacrylate to obtain 2-propionic acid-3-methyl maleic anhydride-polyethylene glycol-poly (2-isopropylamino) ethyl methacrylate. 2-propionic acid-3-methyl maleic anhydride having a concentration of 0.4mmol and a mass of 7.4mg was placed in chloroform having a volume of 20ml, oxalyl chloride having a concentration of 8mmol and a volume of 2ml and dimethylformamide having a volume of 10. Mu.L were added thereto, placed under ice, and stirred sufficiently for 2 hours. After that, a chloroform solution (20 ml) containing hydroxy-polyethylene glycol-poly (2-isopropylamino) methacrylic acid ethyl ester (0.53 g,0.04 mmol) and 4-dimethylaminopyridine DMAP (2.4 mg,0.02 mmol) were added thereto, and the mixture was magnetically stirred at room temperature for 24 hours to obtain a reaction solution. And (3) fully washing the obtained solution with saturated ammonium chloride solution, collecting an organic phase of the solution, filtering, concentrating by rotary evaporation, and finally drying in vacuum to obtain a final product. The CDM-PEG-PDPA synthesis route is shown in FIG. 4.

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Example 1

Rare mutations in the ZNF98 gene are key causative factors of VaMs

(1) Whole exome library preparation and second generation sequencing technique detection

Blood samples were collected from the department of medical care for women and children in Guangzhou city, university of Zhongshan, which included 615 healthy persons as a control group and 334 VaMs patients as an experimental group, and the steps of detailed total exon depth sequencing, data screening quality control and systematic analysis of candidate genes associated with disease were shown in FIG. 5. Whole genome DNA was extracted from venous blood samples (5 ml) of experimental and control group patients using MagPure Buffy Coat DNA Midi KF kit (guangzhou mei biotechnology limited) according to the instructions. The genome DNA is cut and fragmented by using Segmentase (Shenzhen big Gene Co., ltd.) to generate a small DNA fragment with the length of 100-500 bp, and the DNA fragment with the length of 280-320 bp is further enriched by magnetic bead separation. Adenine is added to the 3 '-end of the above DNA fragment so as to be linked to a corresponding adaptor receptor of the 3' -end having thymine, and the DNA fragment is amplified by a polymerase chain reaction mediated by ligand ligation and purified for preparing a whole exome library. The library was subjected to microarray hybridization enrichment using MGIEasy Exome Capture V Probe Set (Shenzhen megagene Co., ltd.) followed by elution and post-capture amplification. Finally, the library products were analyzed in an Agilent 2100 Bioanalyzer to determine the level of enrichment, and then sent to a MGIseq-2000 platform instrument for Huada genes for detection by the "modified-end 100" sequencing strategy line second generation sequencing technique.

(2) Screening quality control of gene mutation sequence data

According to the screening quality control standard issued by NCBI database (Build 37), the sequencing result data is adjusted by using Burrows Wheeler Aligner with reference to the related gene sequence of the human genome (hg 19) to obtain clean data (clean reads) to be analyzed. The repetitive fragments generated by PCR amplification were removed using a picard tool, gene sequence data for local insertion and deletion mutations were rearranged, base mass fractions were calibrated, and gene combination mutation variation was adjusted using GATK HaplotypeCaller. Wherein, the gene mutation sequence data with poor quality are screened out by the following two methods: (a) Screening using the GATK toolkit recommended default dataset and variable quality control score recalibration (Variant Quality Score Recalibration (VQSR)) method of parameters; (b) Deletion of low quality sequencing results and gene mutation sequence data using vcftools software analysis, the relevant parameters were set as follows: -remove-indexes-remove-filtered-geno-all-minGQ 20-minDP 30-mac 3-hwe 0.0001-max-means DP 500-min-legs 2-max-legs 2. The parameter (max-missing-count) was set to 10% of the total number of samples, and the mutation variation of the deletion genotype in 10% or more of the samples was removed.

(3) Principal component and affinity correlation analysis between individuals

Principal component analysis (Principal component analysis, PCA) was performed using gcta64 based on all genetic mutation sites in the han population in the 1000 genome database, and the screening criteria were defined as having an average of the first and second principal components within three standard deviations. 5 outliers were screened out by the method described above and the outlier was culled out (FIG. 6A). Since extreme variability (heterozygosity) between individuals can be caused by DNA contamination or high levels of near-breeding, in order to reduce errors in the analysis of experimental results caused by the above factors, the heterozygosity of the data was calculated using plink affinity analysis, and samples with F coefficients within three standard deviations of the overall population average were retained for further analysis. Meanwhile, plink relatedness analysis was also used to evaluate the correlation between individuals in the experimental group and the control group, and by defining "rel-cutoff" as 0.125, sample data having primary and secondary relatedness could be deleted. The screening method described above removed sequencing results from 61 individuals from the experimental and control groups (fig. 6B).

(4) Analysis of single mutation variation and correlation of whole Gene with disease

Rare mutation sites of the relevant genes were screened from the 1000 genome east asian population, exome Aggregation Consortium (ExAC), ESP6500, dbsnp414 and Genome Aggregation Database (gnomAD) databases, a few allele-frequency less than 1% of the mutation was defined as rare mutation, and correlation between rare mutation of the genes and VaMs was detected using KGGseq software, and Bonferroni corrected exome-wide significance threshold was defined as P-value equal to 0.05/(2 tests× 100252 varians), and the results are shown in fig. 7.

Meanwhile, SNP-set (Sequence) Kernel Association Test (SKAT) analysis was used to detect associations between rare mutations in a set of genes and a binary phenotype (phenotypic classification). The SKAT analysis is performed by using a SKAT module built in KGGseq software, wherein the relevant parameters of the software are set as follows:

-seq-mq 20-seq-fs 60-seq-square 30-vcf-filter-5248.00, vQSRtrancheSNP97.00to99.00-gty-square 20.0-gty-dp 30-gty-af-ref 0.05-gty-af-het0.25-gty-af-alt0.75-hwe-case 0.01-hwe-control 0.01-db-gene, gene-db-scanner-filter-inPASS, VQSRTrancheSNP95.00to, dbfp_known-menu-housing-prediction best-reflection-precursor-filter 1 kgenast 305, dbnp 141, ESns, 5400, pprice, gapeome, db-filter-1-filer-1-kc-signal-1-data-book-1-parts. The Bonferroni corrected exome range significance threshold was defined as a P value equal to 0.05/(2 tests×17427 genes).

The protein-protein interaction network (PPI) encoded by genes associated with the pathogenesis of VaMs was further established using the SKAT assay described above to screen all genes with P values less than 0.05 and the search means of the Retrieval of Interacting Genes/Proteins (STRING) database, the results of which are shown in FIG. 8. Then, the PPI network is divided into different gene clusters by utilizing ClusterOne algorithm, and weighting factors are obtained according to the confidence score of the PPI network, and the construction standard of the gene clusters is defined as at least comprising 5 proteins and the P value of the gene clusters is less than 0.05.

(5) Expression and functional analysis of Gene clusters

The reported 99 genes associated with VaMs and all 18225 genes encoding proteins in the ExAC database were screened out of the network database and defined as 1 gene cluster, respectively, and the loss of function intolerance rate (Probabilities of being intolerant to loss-of-function mutations, pLI) between 9 different gene clusters and the genome of the 2 gene clusters according to the ClusterOne algorithm was calculated, and at the same time, whether there was a difference in pLI value between the different gene clusters was evaluated by using the Student's test, and P value less than 0.05 was defined as having a significant statistical difference. The non-synonymous mutation (non-synonymous variants, NSV) and synonymous mutation (synonymous variants, SV) ratios of the genomes in the different gene clusters were analyzed and compared, and Fisher's exact test was used to evaluate whether there was a difference in NSV/SV between the different gene clusters. Where NSV is a nucleotide variation that can result in amino acid changes, including splicing, deletion, frameshift, insertion, or missense mutation.

The Gene on log (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were enriched for analysis of potential biological functions of the genome in the Gene cluster using the network on-line tool "g: profiler", where P values less than 0.05 were defined as having significant statistical differences. The correlation of genes in the gene clusters with the biological phenotype of the organism was explored using the mouse genome informatics database (Mouse Genome Informatics database, MGI).

Finally, rare mutations of genes were defined as variations with allele frequencies less than 1% and were screened from the 1000 genome east asian population, exAC, ESP6500, dbsnp414 and gnomAD databases. In the full-exon depth sequencing of VaMs blood samples, the screening result of the rare mutation enrichment analysis shows that 704 genes have rare mutations and contain 62703 rare mutation sites, wherein the rare mutation enrichment of ZNF98 gene is most remarkable and the gene with the minimum P value as shown in fig. 8. The research of the invention discovers that the occurrence of domestic VaMs is closely related to rare mutation of genes.

To further investigate the effect of rare mutations in genes on biological function while defining important pathogenic genes for VaMs, protein-protein interaction networks (PPI) were constructed using 704 genes with rare mutations in the experimental group, which showed that the VaMs group contained 593 functional proteins and 1044 interaction links (PPI enrichment P-value equal to 0.3). And the loss of function intolerance (pLI) and non-synonymous mutation (NSV) ratios of the genomes in the different gene clusters were calculated by ClusterOne algorithm, as shown in FIG. 8, showing that pLI scores were significantly higher (P value less than 0.05) and the non-synonymous mutation ratios were lower (P value less than 0.05) for the genes in Cluster 1. The above results show that the genes in Cluster1 are conserved in mutation variation and biological evolution, and that the biological function exerted by the genes is important. We considered that the key causative factor of domestic VaMs was the rare mutation of ZNF98 gene.

Example 2

Method for knocking down ZNF98 gene expression

(1) Construction of siRNA and cell transfection

Transfection with small interfering RNA (siRNA) and Invitrogen (England Life technologies Co., ltd., USA) purchased from Parker's (Rockville, maryland, USA)Agent3000 to establish a ZNF98 knockdown vascular endothelial cell line. Vascular endothelial cells are selected from HUVEC cells and HHSEC cells. The sequences of siRNA are shown as SEQ ID NO. 1-2 (SEQ ID NO.1:5'-GGGAUGUGGCCUUAGAAUUTT-3'; SEQ ID NO.2: 5'-UGACAACUACCCAGAACAATT-3'), and the efficiency of RNA interference is evaluated by qRT-PCR.

(2) Fluorescent real-time quantitative PCR

UsingThe kit extracts total RNA of the cell line, uses All-in-One ^TM qPCR Mix (Guangzhou complex gene Co., ltd.) was used for fluorescent real-time quantitative PCR analysis of gene expression. Detection was performed by Bio-Rad CFX96 and analysis was performed using Bio-Rad management software (Bio-Rad, hercules, calif.). Gene expression levels were normalized to the expression level of housekeeping gene GAPDH and each sample was evaluated three times. The primer sequence of ZNF98 is shown in SEQ ID NO. 3-4 (SEQ ID NO.3:5'-CAGGACCCCTTGGAAGCCTA-3', SEQ ID NO.4:5' -AGAGGCAGCAATACCCACAA-3); the GAPDH primer is shown in SEQ ID No. 5-6 (SEQ ID No.5:5'-GGAGCGAGATCCCTCCAAAAT-3', SEQ ID No.6: 5'-GGCTGTTGTCATACTTCTCATGG-3'). Primers were purchased from guangzhou complex gene limited.

The detection results are shown in FIG. 9.

FIG. 9 shows that ZNF98 expression levels were detected by qPCR (FIG. 9A) in HUVEC cells transiently transfected with ZNF98 targeted siRNA (siRNA-1, siRNA-2) or control siRNA (siNC). ZNF98 expression levels were detected by qPCR (fig. 9B), p <0.01, p <0.0001, student's t test in HHSEC cells transiently transfected with ZNF 98-targeted siRNA (siRNA-1, siRNA-2) or control siRNA (siNC) (both purchased from derivative).

Example 3

Effect of knockdown ZNF98 gene expression on endothelial cell proliferation ability

The effect of endothelial cell proliferation following ZNF98 gene expression in knockdown HUVEC cells and HHSEC cells prepared in example 2 was examined by the following method.

Will contain 8X 10 ³ 100 μl of serum-containing ECM cell suspension of vascular endothelial cells was inoculated in 96-well plates, and the cells were placed in CO at 37deg.C ₂ After culturing for 72 hours in a conventional incubator, the medium in a 96-well plate was blotted, and 10% CCK8 solution was added to each well, and culturing was continued for 2 hours, the absorbance at 450nm was measured with an enzyme-labeled instrument. 5 duplicate wells were set per cell line, with zeroing wells and normal endothelial cell control wells routinely set.

As shown in fig. 9C and 9D, knockdown ZNF98 had no significant effect on cell viability of HUVEC cells (fig. 9C) and HHSEC cells (fig. 9D), and n.s. indicated no statistical significance, student's t test.

Example 4

Effect of knockdown ZNF98 gene expression on endothelial cell migration and tube formation ability

Endothelial cell migration and tube formation following ZNF98 gene expression in knockdown HUVEC cells and HHSEC cells prepared in example 2 were examined according to the following method.

(1) Cell migration assay

Cell migration experiments were performed using a Transwell chamber (Becton Dickinson, franklin Lakes, NJ). Will contain 2X 10 ⁴ 300 μl of serum-free ECM cell suspension of vascular endothelial cells was inoculated in the upper chamber of Transwell, and 700 μl of medium containing 10% fbs was added to the lower chamber. After cells migrating through the membrane were fixed and stained, three fields were counted randomly under an optical microscope.

(2) Cell tube formation assay

Spreading 50 μl Matrigel gel on pre-cooled 96-well plate with thickness of 0.5mm, and placing at 37deg.C CO ₂ And (3) acting for 1h in the incubator, and standing by after the incubator is solidified. The cultured endothelial cells were digested and then treated at 1X 10 ⁴ The wells were resuspended in 100. Mu.l of ECM medium without FBS and inoculated into the 96-well plates described above. After 24h, 3 fields were randomly taken under an optical microscope and the number of capillary lumens and the pseudopodia length were counted.

The results are shown in fig. 9E-J, which show that knockdown ZNF98 inhibited migration of HUVEC and HHSEC cells (fig. 9E), statistical analysis of the number/proportion of vascular endothelial cells that successfully migrated (fig. 9F). Knock-down ZNF98 inhibited the ductal (fig. 9G) and statistical analysis of the number of vascular endothelial cell lumens (No. mes) and pseudopodia length (Tube length) of HUVEC cells (fig. 9H). Knock-down ZNF98 inhibited the ductal (fig. 9I) and statistical analysis of the number of vascular endothelial cell lumens (No. mes) and pseudopodia length (Tube length) of HHSEC cells (fig. 9J), p <0.05, p <0.01, p <0.001,Student's t assays.

Example 5

Knock down of ZNF98 Gene expression on the Convergence of the tail veins of Zebra fish

(1) Knocking down ZNF98 expression in zebra fish embryo

Morpholino modified oligonucleotide sequences purchased from Gene Tools (Oregon, USA) were microinjected (1 nl) during the fertilized eggs 1-4 cells of zebra fish by pressure injector (PLI-100A Pico-liter Microinjector, warner Instruments, USA) to block splice modification of ZNF98 mRNA, which in turn caused deletion mutation of transcript 3 exon of the Gene 36h post fertilization to create ZNF98 knockdown zebra fish. And (5) observing the growth and development of tail vein plexus vessels of the zebra fish larvae under a laser confocal microscope after 72 hours. Among them, EGFP-specifically labeled vascular endothelial cells of transgenic zebra fish (fli 1: EGFP) was purchased from China zebra fish center (China Zebrafish Resource Center, CZRC).

(2) RT-PCR detection

Total RNA from zebra fish larvae 48h after fertilized egg microinjection was purified using TRIzol reagent (Invitrogen, carlsbad, calif.), and reverse transcribed into single stranded cDNA using HiScriptII Reverse Transcriptase reagent (Nannofa Biotechnology Co., ltd.). The amplification was then analyzed by running gel electrophoresis.

(3) Zebra fish angiogenesis observation and analysis

Zebra fish larvae 6h after fertilized egg microinjection were taken and incubated with 1-phenyl-2-thiourea (PTU) purchased from Sigma Aldrich (Sigma Aldrich, inc., usa) to inhibit pigmentation of the larvae. After 72h, the larvae were fixed in 1% low-melting agarose, the growth and development of tail venous plexus vessels of zebra fish larvae were observed by using a laser confocal microscope, and the area of tail venous plexus regions was calculated by detecting with ImageJ software, and the result is shown in fig. 10.

RT-PCR detection results show that after the fertilized eggs of the zebra fish are subjected to microinjection of morpholino modified oligonucleotide sequences, the deletion mutation of the 3 rd exon of the transcript of the ZNF98 gene is successfully caused, and then the ZNF knockdown zebra fish is established (figure 10A).

Fluorescence imaging (fig. 10B) showed that knock-down ZNF98 resulted in growth and neovascularization of the zebra fish tail venous plexus, which was statistically sized (fig. 10C) with p <0.01, student's t-test.

Example 6

Effect of knock-down ZNF98 gene on gene of VEGF signaling pathway

Total cellular RNA was purified using TRIzol reagent (Invitrogen, carlsbad, calif.). RNA-Seq strand-specific libraries were then constructed using each set of samples and sequenced using the NuGEN Ovation RNA-Seq system. The counts for each gene were normalized and then imported into FPKM (number of base fragments per kilobase of transcribed fragment per megabase pair) values. Log of ₂ FC|is not less than 1.5, fold change, p<0.05 was defined as expression differential and normalized using negative and vector controls, respectively. Genes of the bioprocess gene ontology were downloaded from the GO and KEGG pathway database (https:// david. Ncifcrf. Gov /) for analysis of different signal pathways, and the results are shown in FIG. 11.

Screening of differentially expressed genes after HHSEC knock-down ZNF98 using RNA-sequence analysis thermal mapping (FIG. 11A) and volcanic mapping (FIG. 11B) (|log) ₂ (FC)|value>1.0 and P<0.05). (FIG. 11C) VEGF signaling pathway is significantly enriched (in red boxes) in GO and KEGG pathway enrichment assays. (FIG. 11D) qPCR detection of related candidate Gene changes after knockdown of ZNF98 in HHSC cells,/p<0.01，***p<0.001，****p<0.0001,Student's t.

Example 7

ZNF98 gene-loaded targeted small interfering RNA nano-drug

(1) Preparation of alpha-double bond-epsilon-bromopolyethylene glycol (Allys-PEG-Br)

Placing a proper amount of alpha-double bond-epsilon-hydroxyl polyethylene glycol and 2-bromoisobutyl acyl bromide into chloroform, and stirring for 24 hours by a magnetic rotor after the alpha-double bond-epsilon-hydroxyl polyethylene glycol and 2-bromoisobutyl acyl bromide are fully dissolved; the solution obtained is then precipitated twice with cold diethyl ether and washed; and filtering to obtain a solid product, and drying the solid product under vacuum to obtain a final product, wherein the appearance of the final product is white powder. The synthetic route of Allys-PEG-Br is shown in FIG. 1.

(2) Preparation of alpha-hydroxy-epsilon-bromopolyethylene glycol (HO-PEG-Br)

2-mercaptoethanol having a concentration of 20mmol and a volume of 2.17ml and a-double bond-. Epsilon. -bromopolyethylene glycol having a concentration of 2mmol, 7.65g by mass, were sufficiently dissolved in 1, 4-dioxane, AIBN (2, 2' -bis (2-methylpropanenitrile)) having a concentration of 2mmol and a mass of 0.49g by mass was added as a reaction inducer, and reacted sufficiently at a temperature of 70℃for 48 hours. The obtained reaction solution was taken and chloroform was added thereto to obtain a reaction solution. The reaction solution was washed well with saturated ammonium chloride solution, and then the chloroform organic phase solution was concentrated and precipitated with cold diethyl ether. The final product was dried under sufficient vacuum and the final product was pale yellow powder in appearance. The synthetic route for HO-PEG-Br is shown in FIG. 2.

(3) Preparation of hydroxy-polyethylene glycol-poly (2-isopropylamino) ethyl methacrylate (HO-PEG-PDPA)

Alpha-double bond-epsilon-bromine polyethylene glycol with the concentration of 0.05mmol and the mass of 0.13g, cuprous bromide with the concentration of 0.10mmol and the mass of 14.3mg, pentamethyldiethylenetriamine with the concentration of 0.10mmol and the volume of 22 mu L, 2-isopropyl amino ethyl methacrylate with the concentration of 2.5mmol and the volume of 592 mu L, azodiisobutyronitrile with the concentration of 0.01mmol and the mass of 1.6mg are repeatedly frozen and melted for three times in dioxane, and the reactant is placed in a reaction tube at 60 ℃ for sealing reaction for 24 hours. The reaction tube is placed at low temperature to quickly cool down. Then, the product was placed in a dialysis bag at room temperature, the dialysis bag (molecular weight 3.5 kDa) was placed in a large amount of deionized water, and after sufficient dialysis (about 48 hours), the sample was freeze-dried, and the final product was in the form of a white powder. The synthetic route for HO-PEG-PDPA is shown in FIG. 3.

(4) Synthesis of 2-propionic acid-3-methylmaleic anhydride-polyethylene glycol-poly (2-isopropylamino) ethyl methacrylate (CDM-PEG-PDPA)

And (3) acyl chloride 2-propionic acid-3-methyl maleic anhydride, and fully reacting the product with hydroxy-polyethylene glycol-poly (2-isopropylamino) ethyl methacrylate to obtain 2-propionic acid-3-methyl maleic anhydride-polyethylene glycol-poly (2-isopropylamino) ethyl methacrylate. 2-propionic acid-3-methyl maleic anhydride having a concentration of 0.4mmol and a mass of 7.4mg was placed in chloroform having a volume of 20ml, oxalyl chloride having a concentration of 8mmol and a volume of 2ml and dimethylformamide having a volume of 10. Mu.L were added thereto, placed under ice, and stirred sufficiently for 2 hours. After that, a chloroform solution (20 ml) containing hydroxy-polyethylene glycol-poly (2-isopropylamino) methacrylic acid ethyl ester (0.53 g,0.04 mmol) and 4-dimethylaminopyridine DMAP (2.4 mg,0.02 mmol) were added thereto, and the mixture was magnetically stirred at room temperature for 24 hours to obtain a reaction solution. And (3) fully washing the obtained solution with saturated ammonium chloride solution, collecting an organic phase of the solution, filtering, concentrating by rotary evaporation, and finally drying in vacuum to obtain a final product. The CDM-PEG-PDPA synthesis route is shown in FIG. 4. The CDM-PEG-PDPA is the empty nano carrier.

(5) ZNF98 gene-loaded CDM-PEG-PDPA nano-drug for targeting small interfering RNA

siRNA targeting ZNF98 gene (siRNA-1:5 '-GGGAUGUGGCCUUAGAAUUTT-3'; siRNA-2:5 '-UGACAACUACCCAGAACAATT-3') is synthesized in vitro and then dissolved in enzyme-free water to obtain the targeting substance. And (3) dissolving the empty nano-carrier obtained in the step (4) in acetone, dropwise adding enzyme-free water, and stirring on a magnetic stirrer at 600rpm for 4 hours to remove the excessive acetone, thereby obtaining an intermediate carrier. Repeatedly extruding the targeting substance and the intermediate carrier on an extruder for 20 times to obtain the nano drug ZNF98-siRNA@CDM-PEG-PDPA.

And analyzing the morphology and the particle size of the synthesized nano-drug by using an electron microscope. The results are shown in FIG. 12.

FIG. 12 shows that the shape of the nano-drug is spherical (12A) and the particle size is 90nm (12B).

Example 8

Mouse VaMs model construction and nano drug ZNF98-siRNA@CDM-PEG-PDPA in-vivo curative effect detection

By expressing the full-length Tie2-wt sum, respectivelyA retrovirus of Tie2-L914F (purchased from a derivative) infects HUVEC cells to give HUVEC-Tie2-wt and HUVEC-Tie2-L914F. After detecting increased phosphorylation of the TIE2 protein in HUVEC-Tie2-L914F, two groups of cells were incubated at 2.5X10 ⁶ 200 μl was suspended in matrigel, and then both groups of cells were injected into both sides of abdomen of 6-7 week old male athymic C57BL/6J mice (Tie 2-wt is control group) respectively and fed conventionally. HUVEC-Tie2-L914F mice from group C57BL/6J could form abdominal vascularized tissue and vascular malformation lesions at day 14 post-injection.

Injecting nano drug ZNF98-siRNA@CDM-PEG-PDPA into the C57BL/6J vascular malformation mouse model by tail vein injection, taking vascular malformation tissue of the mouse VaMs model at a specific time point, fixing for 48 hours by 4% paraformaldehyde, dehydrating and embedding paraffin. The wax block was cut into 5 μm sections by a microtome, and the sections were stained with immunohistochemical antibodies such as ZNF98, VEGF, TUNEL, etc., and after staining, the sections were photographed under a microscope after sealing with a neutral resin, and observed. The method is characterized in that the phenotype change of the vascular malformation tissues of mice grouped by treatment of different nano drugs ZNF98-siRNA@CDM-PEG-PDPA and the difference of the morphology of main organs are studied by hematoxylin-eosin staining and immunohistochemical plasma methods, the liver and kidney functions of the mice in each group are detected by serology at each time point, and the curative effect and the safety of the nano drugs in vivo use are primarily evaluated.

Taking a VaMs model mouse successfully injected with nano drug ZNF98-siRNA@CDM-PEG-PDPA through tail vein at a specific time point, taking a subxiphoid middle incision, exposing the abdominal cavity, turning over the mesentery and the small intestine and freeing portal vein hepatic portal segments, using a 24G vein indwelling needle to puncture the portal vein, slowly injecting a nonionic isotonic iodine contrast agent, and acquiring portal vein and branch radiography images thereof by using Digital Subtraction Angiography (DSA).

DSA contrast (FIG. 13) shows that injection of the nano-drug ZNF98-siRNA@CDM-PEG-PDPA can effectively reduce abnormal portal vein and branches thereof of a VaMs model mouse, and contrast shows that most portal veins and branches thereof are normal.

As shown by the experimental examples, the invention provides application of knocking down the expression of the ZNF98 gene in cells in preparing medicaments for treating vascular malformations. According to the invention, through deep sequencing of the blood sample of a VaMs patient, the ZNF98 gene is subjected to rare mutation and has related mutation sites, and the change of the expression level of ZNF98 in human umbilical vein endothelial cells and human hepatic sinus gland endothelial cells is closely related to the growth and development of blood vessels. ZNF98 can mediate vascular endothelial cell migration and altered tubular function through downstream VEGF pathways leading to the occurrence of VaMs. The effect of specifically inhibiting or knocking out ZNF98 genes is realized by preparing a nano drug ZNF98-siRNA@CDM-PEG-PDPA carrying ZNF98, and the research is expected to provide theoretical basis and experimental basis for diagnosis and treatment of VaMs, and can be used as a drug for treating and preventing the VaMs.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (10)

1. Use of knockdown of ZNF98 gene expression in cells in the manufacture of a medicament for the prevention and/or treatment of vascular malformations.

2. The use according to claim 1, wherein the cells are endothelial cells;

the endothelial cells are umbilical vein endothelial cells or hepatic sinus gland endothelial cells.

3. The use according to claim 2, wherein knocking down the expression of ZNF98 gene in liver sinusoidal gland endothelial cells causes an alteration of the gene in VEGF signaling pathway.

4. The use according to any one of claims 1 to 3, wherein the substance for knocking down ZNF98 gene expression in the cell is a small interfering RNA, dsRNA, microrna or antisense nucleic acid.

5. The application of knocking down the expression of ZNF98 gene in cells in preparing medicines for inhibiting the expression of ZNF98 or VEGF channel related genes.

6. A kit for preventing and/or treating vascular malformations, comprising a substance for knocking down ZNF98 gene expression in a cell.

7. The kit of claim 6, wherein the substance that knocks down ZNF98 gene expression in a cell is a small interfering RNA, dsRNA, microrna, or antisense nucleic acid.

8. A nano-drug for preventing and/or treating vascular malformations, which is characterized by comprising a substance for knocking down ZNF98 gene expression in cells and an empty nano-carrier;

the substances for knocking down ZNF98 gene expression in the cells are small interfering RNA, dsRNA, micro RNA or antisense nucleic acid;

the empty nano-carrier is CDM-PEG-PDPA.

9. A method for preparing a nano-drug for preventing and/or treating vascular malformations, which is characterized by comprising the following steps:

(1) Mixing a substance for knocking down ZNF98 gene expression in cells with water to obtain a target substance;

(2) Mixing the empty nano carrier with acetone to obtain a mixture, and mixing and stirring the mixture with water for 3-5 hours to obtain an intermediate carrier;

(3) And mixing the targeting substance with the intermediate carrier, and extruding to obtain the nano-drug.

10. The method according to claim 9, wherein the rotational speed of the mixing and stirring in the step (2) is 500 to 700rpm;

the number of times of extrusion in the step (3) is 18-22.