The Relationship Between ALK1 Gene Mutation And HHT And Its Intervention

Objective : In this study,we will deeply explore the correlation between NM₀00020.2（ACVRL1）: c.room2398 G > A and the pathogenesis of HHT,and whether has-mi R-188a-3p and has-mi R-6733-3p,has-mi R-199-5p change the binding to ALK-1 through the mutation of An in rs2293092,thus affecting the protein expression of ALK-1,and the possibility of accurate treatment of HHT through micro RNA.Methods:（1）Using pEZX-FR02 luciferase reporter gene plasmid system as a tool,two reporter plasmids,p EZX-rs2293092-G（wild type）and p EZX-rs2293092-A（mutant type）,were constructed and transfected into HEK-293 T cells.The fluorescence was detected by reporter gene detection kit.（2）mi RNA has-mi R-199-5p was found through bioinformatics online software SNPinfo and micro RNA.org.The homozygous mutation of has-mi R-188a-3p and has-mi R-6733-3p with G>A at rs2293092 site may lead to abnormal binding of m RNA and micro RNA（mi RNA）.After co-transfection into HEK-293 T cells,the fluorescence was detected by reporter gene detection kit.（3）Silencing ALK1 gene by si RNA.After transfection of si RNA-NC and si RNA-ALK1 into HUVEC,48 respectively,RNA,was extracted from cells and their changes were detected by real-time fluorescence quantitative PCR.（4）Silencing ALK1 gene by si RNA.Si RNA-NC and si RNA-ALK1 were transfected into cells respectively,and cell tube formation test and CCK8 cell proliferation test were carried out.Results:（1）Dual luciferase gene report assay showed that NM₀00020.2（ACVRL1）:c.*2398G>A,ALK1 expression was decreased.（2）mi RNA has-mi R-199-5p,has-mi R-188a-3p and has-mi R-6733-3p combine with mutant sites to reduce the expression of ALK-1.（3）The expression of ALK1 has an important effect on the tube formation and proliferation of endothelial cells.Conclusion:（1）The possible new pathogenic single nucleotide polymorphism（SNP）locus rs2293092 in ALK-1 reduces the expression of ALK-1.（2）mi RNA has-mi R-199-5p,has-mi R-188a-3p and has-mi R-6733-3p combine with mutant sites to reduce the expression of ALK-1.