| The anterior segment refers to the lens and all ocular tissues in the anterior portion of the lens,including the cornea,ciliary muscle,iris,conjunctiva,and one-third of the vitreous body.The abnormalities of the anterior segment mainly include corneal opacity,heterotopic pupil,and adhesion between the cornea and iris.A single base mutation（+57>C）in the seed region of micro RNA-184（miR-184）contributed to EDICT syndrome,which is an autosomal dominant disorder,mainly manifested by endothelial dystrophy,iris hypoplasia,cataract,and corneal stroma thinning.EDICT syndrome was first discovered in a Swedish family,where family members often suffer from both cataracts and corneal adhesions,resulting in the corneal stroma thinning,and the severity of the disease varies from individual to individual.Later,a single base mutation（+57>C）in the seed region of miR-184 was also found in a family from Northern Ireland,and the affected family members also developed cataract.However,unlike the Swedish family,the affected family members developed symptoms of conical corneas.Currently,the mechanism of miR-184 in the anterior segment dysplasia is unclear.Therefore,constructing miR-184 animal model will help us understand the role of miR-184 in the development of anterior segment structure,clarify the molecular mechanism of miR-184 mutation in the anterior segment dysplasia,and provide a theoretical basis for the research of targeted therapeutic drugs.The laboratory used CRISPR/Cas9 technology to simultaneously knock out two genes,dre-miR-184-1and dre-miR-184-2,which produce mature miR-184 in zebrafish,and successfully constructed miR-184knockout zebrafish.The deletion of mature miR-184 in zebrafish was determined using Real Time PCR.A simple analysis of the phenotype was conducted in the early stage,but its phenotype still needs further clarification and molecular mechanisms need to be explored.We previously conducted a simple analysis of the phenotype,so the phenotype still needs to be reserached and the molecular mechanism needs to be further explored.To confirm the microphthalmic phenotype of miR-184-/-zebrafish,we took live photos and counted eye sizes of 7 dpf,14 dpf,and adult wild type and miR-184-/-zebrafish.At the same time,paraffin section H&E staining was performed on the eyes of wild type and miR-184-/-zebrafish at the ages of 1 and2months.The results showed that compared with wild type zebrafish,miR-184-/-zebrafish eyes began to develop microphthalmia at the age of 2 months,and the difference became more significant with age.In order to determine whether the cornea of zebrafish showed abnormalities after knocking out miR-184,we used AS-OCT and macro magnification microscopy to observe the anterior segment structure of wild-type and miR-184-/-zebrafish in vivo.AS-OCT and in vivo observation results showed that compared with wild-type zebrafish,the lens pellucidity of miR-184-/-zebrafish decreased,but no significant corneal anterior dilation,conical cornea,or iris abnormalities were observed.We compared the thickness of miR-184-/-zebrafish at 2.5 months,5 months,and 12 months of age with that of wild-type corneal epithelial cells and stromal layer,and found no significant abnormalities.This indicates that knocking out miR-184does not affect the corneal structure and development of zebrafish.To confirm the cataract phenotype of miR-184-/-zebrafish,we observed the lens of wild type and miR-184-/-zebrafish using slit lamps for 12 months.The results showed that the lens of miR-184-/-zebrafish became turbid compared to the wild type.Lens grid imaging also indicates that knocking out miR-184cause cataract in zebrafish.Our further study found that there was no abnormality in the degradation of nuclei and mitochondria in miR-184-/-zebrafish’s lens after 3 days,indicating that the lens terminal differentiation of miR-184-/-zebrafish was normal.We found thatγ-crystallin of miR-184-/-zebrafish abnormally accumulated.And the disordered arrangement of lens skeleton protein F-actin may bethe cause of cataract in miR-184-/-zebrafish.Further research found that the expression of p21,independent of p53,in the optic cup and lens was significantly upregulated.The expression of fibrosis genes in the lens of zebrafish miR-184-/-is upregulated,and the lens has a tendency to fibrosis.Inhibition of p21 activity can significantly downregulate the expression of fibrosis genes.The results suggest that the high expression of p21 may be another reason for the occurrence of cataract in miR-184-/-zebrafish.In order to investigate the downstream genes regulated by miR-184,we conducted transcriptome analysis and dual luciferase reporter assay screening of wild-type and miR-184-/-zebrafish eyes.The transcriptome results showed that after miR-184 was knocked out,the expression levels of 466 genes in zebrafish eyes increased and 489 genes decreased,including the expression levels of genes related to cell growth and apoptosis decreased,and the expression of genes related to lens protein increased.q RT-PCR was used to verify the results of the transcriptome.In the eyes of miR-184-/-zebrafish,the expression levels of the four transcription factors Hsf4,Sox9a,Ctcf and Smad6a that can inhibit the expression of p21 decreased.At the same time,the direct downstream targets Mybl1,Atp1a3a,Phox2bb,Ampd2a,Nck2a,Akap1b,C1orf174 and Ppp6r2b of miR-184 were screened out by the Luciferase Reporter Assay.Using 3-month-old miR-184-/-zebrafish lens and optic cup RNA for validation,it was found that Atp1a3a,Nck2a,Akap1b,and Ampd2a were upregulated in miR-184-/-zebrafish lens expression,and there was no significant difference in the expression of each gene in optic cup between the two.In conclusion,our results indicate that miR-184 participates in the development of anterior segment by regulating p21 expression.Our findings help us understand the role of miR-184 in the development of anterior segment,and provide a new thinking direction for clarifying the pathogenic mechanism of anterior segment dysplasia caused by miR-184 mutations. |
PDF Full Text Request