Micro-132-3p Regulates Oxidative Stress By Targeting FOXO3 In H-Constant Spring α-thalassemia

Objective Thalassemia is one of the most common monogenic genetic diseases in human beings.The incidence of α-thalassemia is high,and the population base is large.Some patients need lifelong blood transfusion and are prone to secondary complications,which seriously affect the quality of life of the patients,it causes a great economic burden to the family and society.it is of great significance to study the pathogenesis and clinical heterogeneity of α-thalassemia.Oxidative stress disorder is one of the important factors affecting the severity of clinical symptoms of thalassemia.The purpose of this study was to explore the core genes and mi RNA related to the oxidative stress of H-CS α-thalassemia by RNA and mi RNA transcriptome sequencing,so as to explore the mechanism of α-thalassemia and other types of thalassemia and to find effective ways to alleviate and treat thalassemia.Methods In this study,high-throughput RNA transcriptome sequencing and mi RNA transcriptome sequencing were used to detect the transcriptional changes of H-CS α-thalassemia(H-CS group,n = 6)and healthy controls(group N,n = 3).To explore the potential function of differentially expressed genes and mi RNA in H-CS α-thalassemia,Bioinformatics analysis methods such as GO enrichment analysis and KEGG enrichment analysis were used to analyze the sequencing data,and RT-q PCR method was used to verify the differentially expressed genes and mi RNA.In the cellular mechanism experiment,fresh anticoagulant blood was taken from patients with H-CS α-thalassemia and healthy controls,and the levels of ROS and MDA in peripheral blood of the two groups were detected by flow cytometry and colorimetry to explore the level of oxidative stress in patients with H-CS α-thalassemia.The oxidative stress related gene set was obtained in the "Gene Cards" database with "oxidative" as the key word,and intersected with the differentially expressed gene set obtained by RNA sequencing to find the possible target genes.The population sample size was expanded and verified by the method of RT-q PCR.The targeting relationship between mi RNA and target gene was predicted by bioinformatics technology,the targeting relationship was verified by double luciferase experiment,the protein level of target gene after overexpression of mi RNA was detected by Western blot method,and the expression level of mi RNA in HCS α-thalassemia patients was verified by RT-q PCR after RNA extraction.The model of overexpression and inhibition of mi RNA expression in K562 cells was constructed.The level of ROS was detected by flow cytometry,and the expression levels of mi RNA,target genes and oxidative stress-related marker genes were detected by RT-q PCR.Results A total of 1585 differentially expressed genes and 358 mi RNAs were screened in this study.Based on the analysis of differential genes combined with Gene Cards database,8 genes were found to be significantly related to the occurrence and development of thalassemia.GO enrichment analysis was carried out on the differential genes,which showed that the differential genes were mainly enriched in the cellular components such as nucleus,cytoplasm and nucleolus,and participated in the molecular functions such as ubiquitin binding and ubiquitin-like protein binding.KEGG enrichment analysis showed that the differential genes were mainly enriched in ubiquitin-mediated protein hydrolysis,FOXO signal pathway and so on.5 genes and 7 mi RNAs with significant differences in sequencing samples were verified by RT-q PCR clinical samples,and the results were consistent with the change trend of expression in sequencing data.The results of flow cytometry showed that the level of ROS in patients with H-CS α-thalassemia was higher than that in healthy controls,and the results of colorimetric test showed that the expression of MDA in patients with H-CS α-thalassemia was higher than that in healthy controls.Bioinformatics analysis showed that 24 genes may be involved in the regulation of oxidative stress of H-CS α-thalassemia.After expanding the sample size of the population,the results of RT-q PCR method showed that the expression of FOXO3 was significantly higher in patients with H-CS α-thalassemia.The target gene prediction database of mi RNA and bioinformatics showed that 7 bases in the coding region of mi R-132-3P were complementary to those in FOXO33’UTR region.Double luciferase reporter gene assay showed that there was a targeted relationship between mi R-132-3P and FOXO3 genes,and the western blot assay showed that mi R-132-3p could inhibit the expression of FOXO3 protein.The RT-q PCR results showed that overexpression and inhibition of mi R-132-3P were successful.Further examination of the expression levels of FOXO3,CAT and SOD1 showed that the expression levels of FOXO3,CAT and SOD1 decreased after overexpression of mi R-132-3P.After inhibition of mi R-132-3P,the expression levels of FOXO3,CAT and SOD1 were up-regulated.The results of flow cytometry showed that the level of ROS in K562 cells increased after overexpression of mi R-132-3P,and decreased after inhibition of mi R-132-3P.Conclusion our study preliminarily explored the change level of H-CS α-thalassemia reticulocyte transcriptome.According to the results of sequencing analysis,we speculate that FOXO signal pathway may play a role in the pathogenesis and clinical phenotype of H-CS α-thalassemia.The level of oxidative stress in erythrocytes of patients with H-CS α-thalassemia is increased,and the increase of FOXO3 may be related to the regulation of oxidative stress in patients with H-CS α-thalassemia.mi R-132-3P may affect the expression of ROS and reactive oxygen scavenging enzymes SOD1 and CAT through miR-132-3P/FOXO3 axis.