The Effect And Mechanism Of CircLRBA On The Liver Regeneration After 2/3 Partial Hepatectomy In Mice

Objective:As the most substantial organ in the body,the liver can perform multiple biological functions,including the production of plasma proteins and clotting factors,the secretion of bile,the balance process of sugar metabolism and lipid metabolism,and the transformation and elimination of biotoxins.In addition,the remarkable regenerative capacity of the liver is also the important characteristic that distinguishes it from other organs,allowing the liver to restore the structural and functional integrity in a short time after being injured.For decades,the preliminary breakthroughs had been made in the exploration of liver regeneration,while the role of non-codingRNA in the process of liver regeneration is still unclear.CircularRNA,as an emerging non-codingRNA widely distributed in eukaryotic cells,involved in the growth of human organs,the metabolism of body,and the progression of diseases.Although the effect of several circRNAs in the development of multiple pathologies of the liver(liver fibrosis,liver cancer)has been identified and recognized,the regulatory role of circRNA during the process of liver regeneration is not fully understood.Lipopolysaccharide-responsive beige-like anchor gene(LRBA)had already been confirmed to promote the proliferation of a variety of tumor cells,but the biological function of its circular molecule circLRBA has not yet been reported,especially in the pathophysiology process of liver.The main purpose of this study is to explore the biological functions and regulatory mechanisms of circLRBA in the process of liver regeneration,which will provide more theoretical data and experimental information for further revealing the molecular network of liver regeneration.Materials and Methods:1.We first conducted a 2/3 partial hepatectomy model in wild-type mice,removed and collected the liver tissues at different time points after resection,detected the proportion of Brd U positive cells of liver tissues at different time points by immunohistochemical staining.Then we validated and analyzed the expression trend of 8 circRNAs(circLRBA1-8)produced by reverse splicing after transcription of LRBA gene at different time points by q RT-PCR,and selected the optimal sequence of circLRBA whose expression tendency was compatible with the liver regeneration process for further research.By designing and synthesizing the specific shRNA and overexpression lentivirus,remarkably knocked down or improved the expression level of circLRBA in immortalized mouse hepatocyte line(AML12)and mouse hepatoma cell line(Hepa1-6),then the CCK-8proliferation experiment,EDU proliferation experiment and cell cycle experiment verified the regulatory effect of circLRBA in hepatocyte proliferation and cell cycle.Finally,the western blot was performed to verify the expression level of cell cycling proteins under different conditions(circLRBA knocked down or overexpressed)in abovementioned two cell lines.2.The model of circLRBA be knocked down in vivo was constructed by designing and synthesizing the adeno-associated virus carrying shRNA sequence and negative control virus through the tail vein of mice,then a 2/3 partial hepatectomy was performed in the successfully constructed mice.Liver tissues were collected at several significant time points(0h、24h、36h、48h、120h)after surgery.Then we detected the expression level of circLRBA in experimental group and control group through q RT-PCR at above time points.In addition,the immunohistochemical staining was used to detect the positive cells ratio of Brd U,Ki67 and p H3S10 in the two groups,and the residual liver/weight ratio of the two groups were calculated,serum biochemistry test the alanine aminotransferase(ALT),aspartate aminotransferase(AST)and total bilirubin(TB)levels at different time points of the two groups.Finally,western blot was used to verify the expression levels of cell cycling proteins at different time points in experimental group and control group.3.Selecting the AML12 cell line which circLRBA was stable knocked down and the control cell,as well as the liver tissues which circLRBA was stable knocked down removed at 36 h and the control liver tissue removed at 36 h after2/3 liver resection forRNA sequencing.Screening out the differentially expressed genes among the genes detected by sequencing,and the GO function enrichment,KEGG pathway enrichment were carried out to identify the function of them.Then the protein interaction network(PPI)was also established.4.Bioinformatics analysis predicting the microRNAs that circLRBA could combine through “sponge manner”,selecting the miRNAs related to liver regeneration and verified them by the dual luciferase experiment.Synthesizing the inhibitor of micoRNA to inhibit the expression of micrRNAs,the vitro cell proliferation experiments were performed to verify the biological function of the microRNAs.TheRNApull-down experiment was performed to enrich the proteins that circLRBA may combine.Mass spectrometry analysis was to screen the optimalRNA binding protein,and their mutual relationship was validated reversely through theRNA immunoprecipitation(RIP),Co-Immunoprecipitation(Co-Ip)and immunofluorescence analysis.Results:1.Brd U staining at different time points found that the positive cells ratio was at the peak on postoperative 36 h after 2/3 partial hepatectomy in wild-type mice,the process of DNA replication and mitosis were active.By screening the 8circRNA molecular produced by transcription of LRBA gene,it was found that the expression tendency of the mmu-circ-0010831 sequence during liver regeneration in mice was optimal according to Brd U staining,similar to the expression tendency of the LRBA gene,both of them showed the tendency to promote the liver regeneration.The vitro experiments on AML12 and Hepa1-6 cell lines showed that cell proliferation were slowed down,cell cycle were blocked,and the proportion of dividing cells were significantly reduced when circLRBA were knocked down;while it can be observed the improved cell proliferation rate,accelerated cell cycle,and increased proportion of dividing cells in above cell lines when circLRBA was overexpressed.The expression level of cell cycling proteins cyclin A2/cyclin E1/cyclin D1/CDK2/CDK4/p H3S10 were reduced when the circLRBA were knocked down in the AML12 and Hepa1-6 cells compared to that of the control group;while the corresponding cycling proteins expression levels were significantly improved compared to the control group when the circLRBA were overexpressed.2.The residual liver/weight ratio at 24 h,36h and 48 h after 2/3 PHx in the group of circLRBA knocked down were significantly lower than that of the control group,while the serum biochemistry(ALT,AST,total bilirubin)levels showed significantly increased;immunohistochemical staining found that the positive hepatocyte cell ratios of three mitotic indicators(Brd U,Ki67,p H3S10)at liver tissues of 24 h,36h and 48 h were significantly reduced when circLRBA was knocked down.And the expression levels of cyclin B1/CDK2/CDK4/p H3S10 in liver tissues of experiment group were also observed a remarkable reduced compared to the negative control group.3.Transcriptome sequencing in AML12 cell samples had screened out 987 differentially expressed genes,the expression level of 456 genes had been significantly up-regulated and 531 genes had been significantly down-regulated;535 differential expressed genes were detected in liver tissue samples,of which the expression level of 292 were up-regulated,and 243 were down-regulated.Through the GO enrichment of differential genes,it was found that terms such as the regulation of the mitotic cycle,the metabolic process of ribonucleotides and the negative regulation of the cell cycle were significantly enriched;the KEGG enrichment of the differential genes found several key liver regeneration relevant signaling pathways(Hippo signaling pathway,p53 signaling pathway,HIF-1signaling pathway,etc.)were significantly enriched;the construction of PPI network further displayed the mutual relationship between cell cycling proteins.4.The results of biosynthesis analysis showed that let-7i,miR-26 a and miR-29 a were potential downstream target miRNAs of circLRBA,the dual luciferase report experiments further indicating that circLRBA could bind to let-7i and miR-29 a.But the vitro cell proliferation experiments showed that let-7i and miR-29 a could not regulate the cell proliferation.ChIRP experiment and mass spectrometry analysis screened out the protein RNF123 that specifically binds to the circLRBA.RNF123 regulating the downstream cyclin-CDK inhibitor p27,the expression level of p27 was verified in cells and tissues that circLRBA were knocked down and over-expressed.Then the RIP、Co-Ip and immunofluorescence technology further confirmed the existence of mutual interaction among circLRBA/RNF123/p27,and theRNA-protein complex was formed based on them to regulate the process of liver regeneration.Conclusion:CircLRBA as a stable non-codingRNA distributed in eukaryotic cytoplasm,it can indirectly regulate the expression of cyclin-dependent kinase inhibitor p27 by combining with E3 ubiquitination ligase RNF123,thereby regulating the hepatocyte cycle and proliferation process,and accelerating the repair of liver structure and function,promoting liver regeneration after liver partial hepatectomy.Therefore,our study contributing to further understand the relationship between circularRNA and liver regeneration,providing the new evidence for in-depth exploration of liver regeneration and even future clinical treatment of liver diseases.