| Background: After most acute and chronic injuries such as resection of the liver,the liver regeneration process will be initiated,and the proliferation of hepatocytes throughout the entire process.A variety of genes and non-codingRNA play an important role in this process,but the specific mechanism is still unclear.Objective: To identify miRNAs and target genes closely related to liver regeneration and proliferation,and to explore their role and mechanism in liver regeneration.Methods:1.Through the establishment of a majority of liver resection models in mice,the application of high-throughput sequencing technology to detect the expression profile of the whole transcriptome,enrichment analysis of differentially expressed mRNA,miRNA,lncRNA,and circRNA,and differentially expressed mRNA,Joint analysis of the interaction of miRNA,lncRNA,and circRNA.Based on the above results,target miRNAs and target genes closely related to cell proliferation with significant differences in expression were screened out.2.We use the dual luciferase report experiment to detect whether the target miRNA is targeted to the target gene;culture mouse liver cells NCTC 1469 in vitro,use plasmid transfection to achieve the target miRNA level overexpression and knockdown,divided into blank control group,Negative control group(NC group)and overexpression/knockdown group.The RT-q PCR method was used to detect the mRNA expression of each group of target genes,the Western blot experiment was used to detect the target gene protein expression of the target miRNA;the CCK-8 method was used to detect the number of cells,and the EDU experiment was used to detect the activity of cell proliferation.Results:1.Differentially expressed mRNA is mainly related to organelle fission,cell nuclear division,chromosome separation,cell cycle,DNA replication,protein-DNA complex construction and assembly,etc.The lncRNA-associated-ceRNA network indicates that lncRNA is involved in the regulation of microRNAs in cancer,alcoholism,systemic lupus erythematosus,viral carcinogenesis,pantothenic acid and coenzyme A biosynthesis and other related signaling pathways by regulating miRNA.The circRNA-associated-ceRNA network shows that circRNA regulates miRNA expression by binding to the corresponding miRNA,thereby participating in related signal pathways such as steroid hormone biosynthesis,retinol metabolism,chemical carcinogenesis,and the mutual conversion of pentose and glucuronic acid.Through the comparison of differential expression,we found that the expression of PDK1 is significantly different and closely related to cell proliferation.Combined analysis of mRNA and miRNA indicated that PDK1 is the regulatory target of miR-34b-5p.2.The results of the dual luciferase reporter gene showed that the overexpression of mmu-miR-34b-5p significantly reduced the activity of PDK1 3’-UTR wild-type luciferase(P<0.05),while PDK1 3’-UTR mutant cells It shows higher luciferase activity.RT-q PCR results showed that the relative expression of PDK1 mRNA in the overexpression group was significantly lower than that of the blank control group and NC group(P<0.05),while the relative expression of PDK1 mRNA in the knockdown group was significantly higher than that of the blank control group and NC group(P<0.05).The results of CCK-8 showed that the number of cells in the overexpression group was significantly lower than that of the blank control group and NC group(P<0.05),while the number of cells in the knockdown group was higher than that of the blank control group and NC group(P<0.05).EDU experiment results: the cell proliferation activity value of the overexpression group was lower than that of the NC group and the control group(P<0.05),and the cell activity of the knockdown group was significantly higher than that of the blank control group and the NC group(P<0.05).Conclusion: This study revealed the expression and enrichment of mRNA,miRNA,circRNA,and lncRNA during liver regeneration,and confirmed that the miR-34b-5p/PDK1 axis can target hepatocyte proliferation.Therefore,inhibiting miR-34b-5p may become an important target in improving liver regeneration. |
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