Application of miR-559 as congenital megacolon diagnostic marker

Abstract

The invention relates to the technical field of gene engineering, and provides application of miR-559 as a congenital megacolon diagnostic marker. The applicant researches and finds that the expression of miR-559 in HSCR is remarkably increased, and invasion and proliferation of SH-SY5Y cells can be remarkably promoted by inhibiting the expression of miR-559; meanwhile, the expression of the miR-559 is in positive correlation with the expression of the M1 type macrophage marker in the narrow section of the HSCR child patient; m1 type polarization of THP-1 cells can be remarkably inhibited by inhibiting expression of the polypeptide. In conclusion, by inhibiting the overexpression of the miR-559, M1 type polarization of macrophages can be effectively inhibited, and the invasion and multiplication capacity of ganglion cells is improved, so that the purpose of reducing the HSCR incidence rate is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and particularly to the application of miR-559 as a diagnostic marker for Hirschsprung's disease. Background Art

[0002] Hirschsprung's disease (HSCR), also known as aganglionosis, is one of the most common congenital digestive tract malformations in children, with an incidence of about 1 / 5000 - 1 / 4000, and the male-to-female ratio is about 4:1. Currently, the specific cause and pathogenesis of HSCR are not clear. The main pathological feature of HSCR is that during the 5th - 7th week of embryonic development of the enteric nervous system (ENS), the proliferation, migration, differentiation, and survival of enteric neural crest-derived cells (ENCCs) are impaired, resulting in the failure of enteric neural crest cells to fully colonize the intestine. The main clinical manifestations of children are abdominal distension, vomiting, intestinal obstruction, and defecation disorders. Currently, the main treatment method is surgical resection of the aganglionic intestinal tract, but postoperative refractory constipation and enterocolitis and other complications are likely to occur again, seriously affecting the quality of life and growth and development of children, and the treatment effect is not satisfactory. Therefore, studying the pathogenesis of HSCR and developing an effective targeted monitoring method are of great significance for the prevention of HSCR and the screening of new therapeutic targets. Summary of the Invention

[0003] The purpose of the present invention is to provide the application of miR-559 as a diagnostic marker for Hirschsprung's disease. Abnormal expression of miRNAs is involved in the pathogenesis of HSCR, but its regulatory mechanism is not clear. Currently, there are still many miRNAs to be discovered in the occurrence and development of HSCR. miRNAs may be potential targets for future HSCR treatment, so it is crucial to identify miRNAs and their target genes. Through bioinformatics analysis and clinical sample detection and analysis, the present invention found that the expression of miR-559 in the stenotic segment intestinal tissue of HSCR children was significantly increased, and inhibiting the expression of miR-559 could significantly weaken the proliferation and invasion ability of SH-SY5Y cells, indicating that miR-559 is related to the pathogenesis of HSCR.

[0004] In order to achieve the above invention purpose, the present invention provides the following technical solutions:

[0005] The present invention provides the application of miR-559 as a diagnostic marker for Hirschsprung's disease.

[0006] The present invention also provides the application of miR-559 in the preparation of products for diagnosing or treating targets of Hirschsprung's disease.

[0007] The applicant found that highly expressed miR-559 can promote the M1 polarization of macrophages, affect the invasion and proliferation of neural crest cells, and ultimately promote the occurrence and development of HSCR. Therefore, on the basis of this research, the present invention intends to clarify the relationship and possible mechanism of action between miR-559 regulating the M1 polarization of macrophages and the occurrence of HSCR by using technical means such as qRT-PCR, Western blot, luciferase reporter gene vector, RNA-seq on cell and tissue samples of HSCR children; and evaluate the possibility of miR-559 as a diagnostic and treatment target through serum samples of HSCR children, which has important basic research and clinical application significance.

[0008] The research objectives of this application are: (1) To explore the relationship between the change in miR-559 expression level and the M1 polarization of macrophages and the occurrence of HSCR. (2) To explore the possible mechanism by which miR-559 regulates the M1 polarization of macrophages and participates in the occurrence and development of HSCR.

[0009] The key scientific problems to be solved in this application are: There are a large number of miRNAs and numerous regulatory targets in the body. At present, the relationship between miRNAs and the occurrence of HSCR is not clear and it is still in the initial stage of research. Macrophage polarization is closely related to the enteric nervous system (ENS). M2 macrophages can promote the differentiation of neural crest cells, the colonization and migration of ganglion cells, and macrophage polarization is regulated by miRNA expression. Through bioinformatics analysis and experimental verification, the applicant found that miR-559 is significantly up-regulated in HSCR and can promote the M1 polarization of macrophages, but the specific mechanism of miR-559 regulating the M1 polarization of macrophages and its relationship with the occurrence of HSCR are not clear. Therefore: (1) This application intends to explore the relationship between miRNAs and the occurrence of HSCR from the perspective of miRNA regulating macrophage polarization; (2) clarify the possible mechanism by which miR-559 regulates the M1 polarization of macrophages and participates in the pathogenesis of HSCR, and explore the possibility of serum miR-559 expression as a diagnostic marker for HSCR.

[0010] The beneficial effects of the present invention are as follows: miRNA (microRNA, MiRNA) can affect the migration and colonization of neural crest cells and ultimately affect intestinal nerve function by regulating the M1 polarization of intestinal macrophages. The applicant's research found that reducing the expression level of miR-559 in HSCR can improve the invasion and proliferation of SH-SY5Y cells; at the same time, the expression of miR-559 is positively correlated with the expression of M1 macrophage markers in the stenotic segments of HSCR children; inhibiting its expression can significantly inhibit the M1 polarization of THP-1 cells. In summary, the applicant speculates that the highly expressed miR-559 promotes the M1 polarization of macrophages, affects the invasion and proliferation of ganglion cells, and ultimately leads to the occurrence of HSCR. The present invention uses qRT-PCR, RNA-seq and other methods at the cellular and clinical levels to clarify the relationship and possible mechanism between miR-559 regulating M1 macrophage polarization and the occurrence of HSCR; and evaluates the possibility of miR-559 as a diagnostic and treatment target for HSCR through the serum samples of HSCR children, which has important basic research and clinical application significance. Description of the Drawings

[0011] Figure 1 is the technical roadmap at the clinical level;

[0012] Figure 2 is the technical roadmap at the cellular level;

[0013] Figure 3 is the expression analysis of miR-559 in HSCR, where (A) is the volcano plot of GSE77296 analysis; (B): the expression of has-miR-559 in HSCR tissues (n = 6); DPBS: dilated proximal bowel segment, dilated segment; NDBS: narrow distal bowel segment, stenotic segment; **P < 0.01;

[0014] Figure 4 is the detection of the proliferation ability of SH-SY5Y cells (400X);

[0015] Figure 5 is the detection of the invasion ability of SH-SY5Y cells (compared with the Control group, **P < 0.01);

[0016] Figure 6For MHCII expression analysis, where (A) is the detection of MHCII expression by IHC (n = 6); (B): analysis of MHCII IOD value; (C): analysis of the correlation between miR-559 and MHCII expression; DPBS: dilated proximal bowel segment, the dilated segment; NDBS: narrow distal bowel segment, the narrow segment; compared with the DPBS group, **P < 0.01;

[0017] Figure 7 For the detection of MHCII expression in THP-1 cells by immunofluorescence. Specific implementation manners

[0018] Features and innovations of this project: Hirschsprung's disease is one of the most common congenital digestive tract malformations in children, seriously affecting the health and quality of life of children. Its pathogenesis is a problem that urgently needs to be solved. Abnormal polarization of macrophages seriously affects the function of enteric ganglion cells; there is a close relationship between macrophage polarization and abnormal expression of miRNAs. At the same time, abnormal expression of miRNAs is related to the pathogenesis of HSCR. However, there are few research reports on the interaction between miRNAs and macrophage polarization. This study innovatively explores the pathogenesis of HSCR from the perspective of macrophage polarization; at the same time, from the change of miRNAs expression, explores the possible mechanism of miR-559 regulating M1-type polarization of macrophages, and explores the possibility of miR-559 expression in serum as a diagnostic marker for children with HSCR. This application intends to confirm the role of miR-559 regulating M1-type polarization of macrophages in the pathogenesis of HSCR. It is not only a good supplement to enrich the miRNA participation in the HSCR regulation network, but also provides a new direction and theoretical basis for finding potential diagnosis and treatment targets.

[0019] The technical solutions provided by the present invention will be described in detail below in conjunction with the embodiments, but they cannot be understood as limiting the protection scope of the present invention.

[0020] Example 1 To explore the relationship between the expression of miR-559 and macrophage M1 polarization in the intestinal tissues of children with HSCR

[0021] (1) Collect the clinical relevant information of children with HSCR, surgically resected intestinal tissues (dilated segment, narrow segment, normal segment), serum and serum samples of the control population. The serum is placed at -80 °C for storage for later use. At the same time, surgically resected colon specimens of children without congenital digestive tract malformations and non-intestinal nerve-related diseases (intestinal tubes at the fistula sites of necrotizing enterocolitis, incarcerated hernia and intussusception) are collected. The expression of miR-559 in the intestinal tissues of children with HSCR is detected by qRT-PCR, and its correlation with the clinical information of HSCR is analyzed. All the included researchers signed the informed consent form and obtained the approval of the Ethics Committee of our hospital.

[0022] a. Inclusion criteria for the disease group: Children with Hirschsprung's disease diagnosed and surgically treated in our hospital; The postoperative pathological results confirmed Hirschsprung's disease; Without other congenital malformations or organic diseases.

[0023] b. Inclusion criteria for the control group: Children diagnosed with non-gastrointestinal diseases in our hospital; Without other congenital malformations or organic diseases.

[0024] c. Exclusion criteria: Preoperative diagnosis of Hirschsprung's disease, and the postoperative pathological result is homologous disease of Hirschsprung's disease; Complicated with other gastrointestinal malformations; Complicated with other organic diseases.

[0025] (2) Detect the expression of M1 macrophage markers (MHCII, CD86) in intestinal tissues (dilated segment, stenotic segment, normal segment) by immunofluorescence; Detect the expression of M1 macrophage markers (iNOS, IL-1β, TNF-α) in intestinal tissues (dilated segment, stenotic segment, normal segment) by qRT-PCR.

[0026] (3) Analyze the correlation between miR-559 expression and macrophage M1 polarization in HSCR by linear correlation analysis.

[0027] (4) Collect serum samples from children with HSCR and children with non-gastrointestinal diseases, detect the expression of miR-559 in the sera of children in the two groups by qRT-PCR, and evaluate the possibility of miR-559 as a diagnostic marker for HSCR.

[0028] Example 2 Explore the possible mechanism of miR-559 promoting macrophage M1 polarization and its effect on enteric neural crest cells

[0029] 1. (1) Commercially available THP-1 cells were used as the NC group, and THP-1 cells were induced into M1 macrophages (THP-1-M1) by stimulation with IFN-γ (50 ng / ml) + LPS (15 ng / ml) for 48 h as the Control group (induction reference: Int J MolSci. 2021; 22(20):11252, Cell Rep. 2021; 37(5):109955, Mediators Inflamm. 2019; 2019:1769374). After the overexpression (OV-miR-559) and interference (sh-miR-559) vectors of miR-559 were transfected into THP-1 cells, the expression of MHCII, CD86 was detected by immunofluorescence and the expression of iNOS, IL-1β, TNF-α was detected by qRT-PCR to evaluate the M1 polarization of THP-1 cells.

[0030] (2) After transferring the overexpression (OV-miR-559) and interference (sh-miR-559) vectors of miR-559 into THP-1 cells using RNA-seq technology, analyze the effects of miR-559 on the transcriptome level of THP-1 cells; use bioinformatics analysis methods (starbase2.0, Targetscan, miRDB) to analyze the potential target genes and possible pathways regulated by miR-559. And detect the expression of potential target genes and possible pathways in the intestinal tissue samples of children with HSCR. Explore the possible mechanism by which miR-559 promotes M1 macrophage polarization.

[0031] (3) After using the conditioned medium of THP-1 cells with altered miR-559 expression to act on SH-SY5Y cells respectively, observe the changes in the proliferation, invasion and apoptosis levels of SH-SY5Y cells through EdU, Transwell and apoptosis experiments; explore the effects of THP-1 cells with altered miR-559 expression on the biological functions of neural crest cells. The clinical-level technical roadmap is shown in Figure 1 , and the cell-level technical roadmap is shown in Figure 2 .

[0032] 2. Key technology description:

[0033] (1) Detect the expression of miR-559 in HSCR intestinal tissue by qRT-PCR, immunofluorescence, etc., and analyze its relationship with M1 macrophage polarization.

[0034] (2) Analyze the possible mechanism by which miR-559 expression regulates M1 macrophage polarization through RNA-seq and dual-luciferase reporter gene vector technology.

[0035] (3) Detect the effects of macrophages with altered miR-559 expression on the biological functions of SH-SY5Y cells by EdU, Transwell, and AnnexinV-FITC / PI double staining.

[0036] 3. Feasibility analysis

[0037] (1) Theoretically: Macrophage polarization is closely related to ENS development and can affect the migration, differentiation and ganglion cell function of intestinal neural crest cells; previous studies have found that altered miRNA expression is related to the progression of HSCR. And miRNA is closely related to M1 macrophage polarization. In the early stage of this application, through bioinformatics analysis and clinical sample detection, it was found that the expression of miR-559 was significantly increased in the stenotic segment of the intestinal tissue of children with HSCR; inhibiting miR-559 expression could significantly promote the proliferation and invasion ability of SH-SY5Y cells, and the experimental results are shown in Figures 3 to 5, it is speculated that miR-559 is related to the occurrence of HSCR. However, the specific relationship between miR-559 expression and HSCR needs to be further clarified. The study also found that overexpression of miR-559 can significantly promote the M1 polarization of THP-1 cells as shown in Figure 7 .

[0038] (2) The applicant found in practice that in children with HSCR, the expression of miR-559 is significantly increased, as shown in Figure 3 ; the change in miR-559 expression is closely related to the M1 polarization of THP-1 cells, as shown in Figure 7 .

[0039] Results and Analysis of Example 3

[0040] The experiment found that the expression of miR-559 was significantly increased in the tissues of children with HSCR. Inhibiting the expression of miR-559 could significantly promote the proliferation and invasion of SH-SY5Y cells; overexpressing miR-559 could significantly promote the M1 polarization of THP-1 cells, and its conditioned medium could inhibit the proliferation of SH-SY5Y cells. Specifically as follows:

[0041] (1) The expression of miR-559 was significantly increased in HSCR tissues. Inhibiting the expression of miR-559 could promote the proliferation and invasion of SH-SY5Y cells.

[0042] a. Through the analysis of the GEO (GSE77296) database, it was found that has-miR-559 was significantly increased in the tissues of children with HSCR ( Figure 3 A). By detecting the intestinal tissues of children with HSCR by qRT-PCR, it was found that the expression of has-miR-559 was significantly increased in the stenotic segment (P < 0.01) ( Figure 3 B).

[0043] b. SH-SY5Y cells were intervened with the overexpression (mimics) and interference fragment (inhibitor) of miR-559 respectively. The results showed that: compared with the Control group, there was no significant difference in the proliferation ability of SH-SY5Y cells in the NC group; compared with the Control group, overexpression of miR-559 could significantly inhibit the proliferation of SH-SY5Y cells, and interference with miR-559 could significantly promote the proliferation of SH-SY5Y cells. As Figure 4 .

[0044] c. SH-SY5Y cells were intervened with the overexpression (mimics) and interference fragment (inhibitor) of miR-559 respectively. The results showed that: compared with the Control group, overexpression of miR-559 could significantly inhibit the invasion of SH-SY5Y cells (P < 0.01), and interference with miR-559 could significantly promote the invasion of SH-SY5Y cells (P < 0.01), asFigure 5 .

[0045] (2) M1 macrophages are significantly elevated in HSCR tissues

[0046] IHC detection found that in the HSCR stenosis segment, the expression of the M1 macrophage marker MHCII was significantly elevated (P < 0.01). Linear correlation analysis found that the expression of MHCII was positively correlated with the expression of miR-559 (P < 0.05), as Figure 6 .

[0047] (3) miR-559 can promote M1 polarization of THP-1 cells

[0048] THP-1 cells were intervened with miR-559 overexpression (mimics) and interference fragments (inhibitor) respectively. The results showed that compared with the Control group, miR-559 overexpression could significantly promote the expression of MHCII in THP-1 cells, and miR-559 inhibition could significantly inhibit the expression of MHCII in THP-1 cells, as Figure 7 .

[0049] Conclusion of Example 4

[0050] miR-559 regulates M1 polarization of macrophages and is involved in the pathogenesis of Hirschsprung's disease.

[0051] This application's research found that miR-559 was significantly elevated in HSCR. Inhibiting its expression could significantly promote the invasion and proliferation of SH-SY5Y cells; at the same time, the expression of miR-559 was positively correlated with the expression of the M1 macrophage marker in the stenosis segment of HSCR children. Inhibiting its expression could significantly inhibit the M1 polarization of THP-1 cells.

[0052] As can be seen from the above embodiments, the present invention provides the application of miR-559 as a diagnostic marker for Hirschsprung's disease. MicroRNA (miRNA) can affect the migration and colonization of neural crest cells and ultimately affect intestinal nerve function by regulating the M1 polarization of intestinal macrophages. The applicant's research found that the expression of miR-559 was significantly increased in HSCR, and inhibiting its expression could significantly promote the invasion and proliferation of SH-SY5Y cells; at the same time, the expression of miR-559 was positively correlated with the expression of M1 macrophage markers in the stenotic segment of HSCR children; inhibiting its expression could significantly inhibit the M1 polarization of THP-1 cells. In summary, inhibiting the overexpression of miR-559 can effectively inhibit the M1 polarization of macrophages, improve the invasion and proliferation ability of ganglion cells, and thus achieve the purpose of reducing the incidence of HSCR. Through cell and clinical levels, the present invention uses qRT-PCR, RNA-seq and other means to clarify the relationship and possible mechanism between miR-559 regulating M1 polarization of macrophages and the occurrence of HSCR; and through the serum samples of HSCR children, it evaluates the possibility of miR-559 as a therapeutic target for HSCR, which has important basic research and clinical application significance.

[0053] The above are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. Application of miR-559 as a diagnostic marker for Hirschsprung's disease.

2. Application of miR-559 in the preparation of products for the diagnosis or treatment target of Hirschsprung's disease.

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