Screening Of MicroRNAs Expression Profile In The Process Of Hepatic Fibrosis

Background:Liver fibrosis is a chronic process of intrahepatic damage-repair response. Variouspathogenic factors, such as inflammation, injury, drugs, and genetic factors, play importantroles in this process. The mixed effect of these pathogenic factors results in a abnormalcomprehensive proliferation of fibrous tissue in liver, the diffused produce and depositionof extracellular matrix (ECM), the imbalance of ECM synthesis and degradation,consequently with secondary liver inflammatory response, and the reversible pathologicalprocess of self-healing. Hepatic fibrosis can damage the structure and function of the livertissue. If this process cannot be arrested, liver fibrosis may progress to cirrhosis and evenend-stage liver disease with poor prognosis (such as liver failure), and then affect thepatient quality of life and prognosis seriously. Thus, study on the mechanism of liverfibrosis can help us to find new molecular diagnostical methods and therapeutic targets.Hepatic stellate cell (HSC), a vitamin A storage cells, locates on the liverperisinusoidal Disse space between sinusoidal endothelial cells and liver cells. During therecent decades, large numbers of studies showed that HSC played a central role in theoccurrence and development of liver fibrosis. The joint action of different cytokines, whichare secreted by the liver cells, platelets and sinusoidal endothelial cells, stimulates theactivation of HSC and makes HSC changing into myofibroblast (MFs). The phenotypicand function is different between HSC and MFs. MFs proliferates itself by paracrine orautocrine. Eventually, collagen and proteoglycan synthesized by MFs lead to the formationof intrahepatic collagen fibers. The previous molecular level studies of liver fibrosisfocused on HSC activation and proliferation, ECM synthesis signaling pathway: such asthe cell cycle, apoptosis pathway, inflammatory factors, and fibrogenic factors. However,so far, there is not a treatment can significantly improve liver fibrosis aimed at the hepaticstellate cell activation.microRNA (miRNA) is a short non-coding RNA (18-26nt). miRNA can regulate itstarget gene expression by binding the specific3'-UTRs sequence and play a pivotal rolein a variety of physiological and pathological processes. Mutations, abnormal expressionand elaboration of miRNA will affect the miRNA function, inhibite the mRNA translationof target genes and then silent genes at the posttranscriptional level. Meanwhile, through a complex network-like control system cells, miRNA could precise adjustment organicdevelopment, differentiation, proliferation, apoptosis, immune regulation and otherphysiological activities. Recent studies indicated that miRNA can regulate cellproliferation, apoptosis, lipid metabolism/fatty acid metabolism and play an importantbiological role during the development of many liver diseases. The understanding ofmiRNA epigenetic mechanism provided us new ideas on studying the molecularmechanisms of liver fibrosis development. It is particularly important to find the keymiRNA and signaling pathways involved in liver fibrosis development process. Previousstudies found that lots of miRNA expressed differentially in the HSC activation andproliferation process in vitro. Some of these miRNA were important regulators in thisprocess. For example, miR-15b/16enhanced the apoptosis rate of aHSC through theregulation of apoptosis-related pathways. Additionally, miR-27a/27b affected lipidmetabolism and promoted HSC activation and proliferation, suggesting the possible role ofmiR-27a/27b in HSC proliferation and apoptosis. Taken together, the biological behaviorof HSC can be regulated by miRNA, thereby affecting the process of liver fibrosis. miRNAare expected to become new promising molecular targets of liver fibrosis. However, untilnow, no article has reported the differential expression of miRNA in liver fibrosis processin vivo. The study of the precise role of miRNA in liver fibrosis in vivo will, not only helpus to understand the mechanism of liver fibrosis, but also help us to identify the newmolecular regulators.The aims of this study were:1) detection of miRNA expression profiling indimethylnitrosamine (DMN)-induced liver fibrosis model,2) screening of miRNAdifferentially expressed in the liver fibrosis process,3) clarification the regulative functionof miRNA, target genes, and relevant signaling pathways by bioinformatics,4) screeningand validation of the precise role of the differentially expressed significant miRNA and itstarget genes. Our experiment was divided into three parts:1) the establishment of liverfibrosis model in rats, grouping the liver tissue according to the liver fibrosis levels,2)screening of miRNA differentially expressed in the liver fibrosis, validation of thesemiRNA by real-time quantitative PCR, and foundation of regulatory networks comprisingmiRNA and its target genes,3) screening of the target genes of differentially expressedmiRNA, and check the role in the the process of liver fibrosis. Part I: the establishment of liver fibrosis model in ratsObjective: Establish liver fibrosis in rats to observe the effact of DMN on the generalsituation of rats and liver histopathology, along with the continuation of the modeling cycle.Success to gain rat liver fibrosis tissues at all levels, as well as the material preparation forfurther miRNA microarray expression profiling studies.Methods: DMN was continuous intraperitoneal injected to SD rats in experimental group.The same dose of saline was continuous intraperitoneal injected to SD rats in control group.The general situation of rats was observed for a period of7days regularly. Some rats weresacrificed to get the liver tissue. HE staining and Masson, the VG special staining wereused to determine the degree of hepatic fibrosis in rats.Results:(1) The general situation of SD rats in experimental group changed pathological.Defecation, body hair, and body weight were lower than the control group.(2) The livervolume of experimental group was lower than the control group. A sense of gravel noduleson the liver surface was showed gradually. Intra-abdominal high pressure signs wasemerged during the later period.(3) HE staining and Masson, the VG special staining: thestructure of hepatic lobule was intact in the control group. No necrotic areas were found.Degeneration and necrosis can be seen in the experimental group. A large number ofcollagen fibers and fibrous septa can be found in the livers gradually. Portal area fibrosisand pseudolobule were showed in the later period. The degree of fibrosis was heavier weekby week.Conclusion: Typical liver fibrosis model can be induced by intraperitoneal injection ofDMN for4weeks in SD rats. The pathological features of this model were consistent withthe characteristics of human liver fibrosis. Part II: screening of miRNA differentially expressed in the liver fibrosisprocess and bioinformatics analysisObjective: To study miRNA differentially expresstion in the liver fibrosis process andverify the relevant signaling pathways, to build a regulatory network comprising miRNAand its target genes. Methods:(1) Liver fibrosis tissues were selected according to liver fibrosis pathologicalclassification. miRNA microarray was used to find out the differentially expressed miRNAat every level of liver fibrosis.(2) Real-time quantitative PCR was used to detect theexpression of some miRNA in different levels of liver fibrosis.(3) GO and relevantmetabolic pathway analysis of miRNA-target genes.(4) microRNA and target generegulatory networks were constructed. The key microRNA and target genes were extractedfrom these regulatory networks.Results:(1) Results of miRNA chip analysis suggested that23miRNA differentiallyexpressed in liver tissue in the development of liver fibrosis.(2) Results of real-timequantitative PCR showed miR-34family and miR-181a were significantly upregulated.This was consistent with the results of miRNA chip analysis. The miR-34family levelscorrelated positively with liver fibrosis levels.(3) GO analysis and pathway analysisshowed that: among the gene function and pathways which these differentially expressedmiRNA and their target genes involved, lipid metabolism/fatty acid metabolism andrelative pathways had the significant enrichment degree.(4) miRNA-Gene-Networkanalysis showed that long chain acyl coenzyme A synthetase family members1(ACSL1)located on the center of the regulatory network. ACSL1was regulated by5miRNA.Conclusion: There were miRNA differentially expressed in liver tissue in the developmentof liver fibrosis. The miR-34family levels correlated positively with liver fibrosis levels.These differentially expressed miRNA were involved in the regulation of lipid/fatty acidmetabolism pathway. ACSL1may be an important regulatory molecule in the process ofliver fibrosis. Part III: Detection of target relationship between miR-34famliy and ACSL1, andthe correlation between ACSL1and liver fibrosis gradeObjective: To screen the intrahepatic tissue-specific candidate target genes of miR-34famliy. To validate the targeted relationship between miR-34famliy and ACSL1. To assessthe ACSL1mRNA and protein expression levels in all levels of liver fibrosis.Methods:(1) Candidate target genes of miR-34famliy were screened according totissue-specific expression.(2) Green fluorescent protein (EGFP) reporting system was used to verified the targeted relationship between miR-34famliy and ACSL1.(3) Real-timequantitative PCR and Western-blot were used to measure ACSL1mRNA levels andprotein expression levels in the process of liver fibrosis.Results:(1) there was a miR-34family specific binding site in the3'-UTR of ACSL1mRNA.(2) Results of the EGFP reporter system showed ACSL1was a target gene ofmiR-34a/34c.(3) Real-time quantitative RT-PCR and Western-blot tests showed thatACSL1mRNA levels and protein expression levels were significantly decreased in thedevelopment of liver fibrosis. This result suggested that ACSL1may be an importantregulatory molecule in the process of liver fibrosis.Conclusion: ACSL1expression levels were correlated negatively with liver fibrosis levels.miR-34a/34c and ACSL1are important regulatory molecules in the process of liverfibrosis.