Regulatory Networks Of MicroRNA In The Ferroptosis Pathway

Objective: Through literature research and analysis,combined with the ferroptosis pathway,key regulatory genes were found,and bioinformatics was used to predict the microRNAs(miRNAs)that could affect the 3'UTR regions of these key genes.The effects of miRNAs on ferroptosis were studied in A549 and HeLa cell lines respectively.The regulatory network of miRNA was drawn according to the results obtained,verifying the reliability of the regulatory network and to clarify the correlation between miRNA and ferroptosis..Method: Refer to the literature and analyze the ferroptosis pathway to obtain genes that have a regulatory role in the occurrence and development of ferroptosis,design primer sequences for the 3'UTR region of the gene,and amplify the 3'UTR region by PCR and construct it into the expression vector PGL-3M.With the help of the bioinformatics analysis website Targetscan,we obtained miRNAs that may interact with the 3'UTR region of key genes,amplified the required miRNAs from the existing miRNA expression library in the laboratory,and carried out dual-luciferase reporter gene analysis in 293 T cells.Preliminary detection was carried out in the experiment,and miRNAs that acted on the 3'UTR region of key genes were screened.They were overexpressed in A549 cells and He La cells by transfection with jet PRIME(?) reagent,and under the action of the ferroptosis inducer RSL3,flow cytometry was used The effect of miRNAs on lipid peroxidation,a biochemical indicator of ferroptosis,was detected by cytometry.Combined with the experimental results,the regulatory network of miRNAs on their target genes was analyzed and summarized in A549 and He La cells.The obtained regulatory network was verified.q RT-PCR and Western blot were used to detect the specific genes regulated by miRNA,and at the same time to detect the changes of miRNA on MDA and cell viability when ferroptosis occurred,to verify the effect of miRNA,and to explore the role of microRNA in ferroptosis signaling.The mechanism at play in the pathway.Result: 1.Through literature research and analysis,12 key regulatory genes in the ferroptosis pathway were obtained,and the 3'UTR regions of 12 key genes were successfully amplified and incorporated into the PGL-3M expression vector.132 miRNAs that may interact with genes were obtained through the Targetscan website,and the required microRNAs were successfully amplified from the existing miRNA expression library.3.The dual-luciferase reporter gene experiment screened 42 miRNAs that acted on the 3'UTR region of key genes.4.The effect of overexpressed miRNAs was verified in A549 and Hela cells respectively,and the changes in lipid peroxidation levels were detected by flow cytometry,and 12 miRNAs that functioned in A549 cells were obtained,and they functioned in He La cells The 11 miRNAs,combined with the genes targeted by miRNAs,were obtained in A549 and He La cells to obtain the regulatory network map of miRNAs for key genes of ferroptosis.5.In-depth research on mi R-9 in the regulatory network of A549 cells and mi R-195 in the regulatory network of Hela cells was conducted.The results showed that mi R-9promoted ferroptosis in A549 cells by targeting the GCH1 gene.mi R-195 produced ferroptosis inhibition in Hela cells by targeting ACSL4.Conclision: In the ferroptosis signaling pathway,miRNA can play a role in the occurrence and development of ferroptosis by affecting the expression of key genes.In different cell lines,miRNA functions in different ways,and the results are also not the same,providing new ideas for the study of ferroptosis.