The Study Of MiR-9a-5p Mediated The Sustained Activation Of Hepatic Stellate Cells In Cirrhosis And Portal Hypertension

Background and objectivePortal hypertension is a syndrome with the main characteristics of the portal system hemodynamics changed. Clinical manifestations are esophageal gastric varices, ascites, splenomegaly in gastrointestinal bleeding, hepatic encephalopathy and hepatorenal syndrome. Hepatic stellate cells activation is the key factor in promoting the occurrence and development of liver cirrhosis with portal hypertension. Hepatic stellate cells regulating blood flow of hepatic sinus have very strong contractility, Being similar to the smooth muscle cells. The mechanism is that hepatic stellate cells secrete ECM and migrate to the liver parenchyma cells, causing the swelling of liver cells and the increase of vascular resistance, resulting in increased portal pressure. MicroRNAs (miRNAs), found in recent year, are small noncoding 22nt RNAs that regulate gene expression by binding to the 3’untranslated region (3’UTR) of mRNAs to cause their degradation or translational repression. In liver fibrosis, miRNAs play an important role in regulating HSCs differentiation, proliferation and apoptosis. However, little is known whether miRNAs play a role in HSCs responding to the portal vein pressure. Inquiring the act of microRNA in the HSC under pressure is available for further verify the mechanisms of portal hypertension and for the evidences to possible treatments of portal hypertension.In the study, HSC pressure-loading apparatus was emploied to screen the pressurization induced miRNAs expression profile by next-generation sequencing(Choose 14 days fully activated HSCs). Among the microRNAs, miR-9a-5p was verified to be importantly upregulated during HSC pressure-loading by qRT-PCR. Our previous study found that 10 MMHG(millimetres of mercury) pressure for 1 h could significantly promote the HSCs proliferation, activation and migration through Integrin-FAK pathway. FAK could also activate downstream signaling molecules including Akt. Therefore, effects of miR-9a-5p in HSC consistent activation were detected. The current study contains three sections:(1) Isolation and identification of primary HSCs; (2) miRNA expression profiles of primary HSCs under pressure overload; (3) miR-9a-5p may involve in HSC activation under the condition of cirrhotic portal hypertension.Method1 Isolation and culturing of primary HSCs The isolated HSCs were fostered in DMEM (Dulbecco’s modified Eagle’s medium), containing 20% FBS,100 U/ml penicillin and 100 mg/ml streptomycin. Primary HSCs kept quiescent for 2 days and became activated completely at 14days. Purity identification of primary HSCs was determined by autofluorescence and immunostaining using antibody against desmin and α-SMA.2 Pressure loading Taking 14 days cultured primary HSCs in the pressure device, respectively connected the nitrogen and hematomanometer, opened the hematomanometer, slowly injected nitrogen, when the pressure rose to 10 mmHg, closed the valve and sealed the container, it could be used in subsequent experiments after loading 1 hour.3 Expression profile and bioinformatics analysis of microRNA in Hepatic stellate cell of pressure action3.1 Put hepatic stellate cells cultured in vitro for 14 days as control group (CON HSC); after 14 days, pressured under 10 mmHg for 1 hour as a pressure group (experimental group, P HSC).3.2 Bioinformatics analysis between different microRNA:Gene ontology (GO) analysis---use GO-Analysis to functional analysis in different microRNA, we found likely enrichment function, looking for correlation of which genes’ functional changing, that the different microRNA might be associated with. Pathway analysis--- based on KEGG database, received a significant enrichment of biological signal path or metabolic pathways, looking for correlation of which which cellular pathways’ changing, that the different microRNA might be associated with.4 Validation of miR-9a-5p in HSC overpressure and hepatic tissue of liver cirrhosis in rats4.1 HSC under pressure, expression test of MiR-9a-5p:applied RT-PCR to detect gene expressive quantity’s changing between MiR-9a-5p in HSC of pressure action and MiR-9a-5p in the normal training HSC.4.2 Expression analysis of MiR-9a-5p in advanced hepatic tissue of liver cirrhosis in rats.4.2.1 CCl4 construction of Liver cirrhosis’ animal model:in CON HSC, intraperitoneal injection of 2ml olive oil twice a week; in P HSC (experimental group), intraperitoneal injection of 40% of 2ml olive oil twice a week. Double of the initial dose, normal water feeding. Eight weeks in total.4.2.2 Applied RT-PCR to detect gene expressive quantity’s changing between MiR-9a-5p in normal hepatic tissue and MiR-9a-5p in liver cirrhosis rats’ hepatic tissue.5 Functional testing of HSC under pressure5.1 Applied RT-PCR to detect Type I collagen and alpha SMA expression in HSC undergo pressure.5.2 Applied Western Blot to detect Type I collagen’s and alpha SMA mRNA’s gene expression in HSC of pressure action.6 Transfection of miRNA mimics and inhibitorsCells were transfected using by ribo FECTTM CP Transfection Kit according to the manufacturer’s instructions. Control miR-9a-5p, miR-9a-5p inhibitor and miR-9a-5p mimic were purchased from Guangzhou RiboBio Co., Ltd. Mirs inhibitor and mirs mimic were transfected into cells at density of 100nM and 50nM, respectively, and collected after 24-48 h.7 Proliferation testing of HSC under pressureAfter transfection of miRNA inhibitors and pressure loadinng(10mmHg, 1h), cells were cultivated in 96-well plates and cultured for 24,48 and 72 h. Cell Counting Kit 8 and ELx800 microplate reader (absorbance at 450 nm) were used for identification.8 Migration testing of HSC under pressureTranswell was used for migration assays experiments. HSCs were transfected and pressure loading(10mmHg, 1h) by described previously. According to density of 5×103cells/well, inoculated 100ul cell suspension of each s mall chamber, put Transwell in the culture plate of 24 hole, blue dyed the upside and bottom cells in membrane of Transwell. After a migration for 24 h, observed the downside cells in vision of 400 times count, average a vision includi ng 50-100 cells, observed five visions. The fundation of the filter was visible for counting the number of cells on each face.9 Searching and verification for target genes of MiR-9a-5p Applied luciferase to report gene detect, it had targeted relationship with Western Blot detecting of MiR-9a-5p and Sirtl.10 Statistical analysis All data are showed as mean±SD of three separated experiments. Student’s t-test was performed when data were consisted of only two groups. One-way ANOVA was performed when three or more groups were compared. The SPSS software, version 16.0, was used for all of the statistical analyses and p value of< 0.05 was considered statistically significant.Results1 Isolation and identification of primary HSCs Freshly isolated living HSCs were small and round and appeared strong refraction. These cells were form uniform and far less than liver cells. After cultured for 2 days, the cells adhered to the dish and presented an elliptical shape, then the visible fat particles. The cells exhibited narrow antenna at Day 7 and fully extended at Day 14 (Figure.lA). HSCs showed a blue-green intrinsic autofluorescence under 328 nm uv irradiation (Figure.1A) and showed positive desmin and a-SMA staining at Day 14 (Figure. 1B), indicating the purity of isolated primary HSCs.2 Expression profile and bioinformatics analysis of microRNA in Hepatic stellate cell of pressure action2.118 of miRNAs (miR-206-3p, miR-210-3p, miR-494-5p, miR-345-3p, miR-592, miR-124-3p, miR-429, miR-34c-5p, miR-34b-5p, miR-34a-3p, miR-450a-3p, miR-199a-5p, miR-222-3p, miR-450a-5p, miR-34a-5p, miR-494-3p, miR-377-5p, miR-9a-5p) were upregulated and the rest (miR-26a-3p, miR-222-5p, miR-335, miR-194-3p, miR-103-1-5p, miR-664-1-5p, miR-188-3p) were downregulated.2.2 Upregulated miRNAs regulated 15 GOs and downregulated miRNAs regulated 15 GOs (Figure.2A). GOs of all miRNAs regulation were associated with cell cycle, negative regulation of fibroblast proliferation, positive regulation of macroautophagy, response to retinoic acid, protein K48-linked deubiquitination, protein K63-linked deubiquitination and negative regulation of gene expression. Meanwhile, miRNAs regulate critical pathway contained RNA transport, focal adhesion, NF-kappa B signaling pathway, cell adhesion, molecules and so on.3 Validation of miR-9a-5p in HSC overpressure and liver cirrhosis rats’hepatic tissue Applied RT-PCR verified that HSC overpressure significantly increased the more expression of miR-9a-5p than normal one, consistent with the results of the next generation sequencing. The rat liver tissues of cirrhosis increased the more expression of miR-9a-5p than normal one.4 miR-9a-5p affects the functions of HSC under pressure Through transfection of miR-9a-5p inhibitor and mimic into day 14 culture-activated HSCs, pressure application for 1 h, the expression of a-SMA and Coll was decreased and increased respectively compared with control-miRNA transfected.5 miR-9a-5p under pressure affects HSC proliferation CCK-8 assay showed that the proliferation rate of HSC of miR-9a-5p inhibitor were restrained upon pressure overload.6 miR-9a-5p under pressure affects HSC migration Transwell assay showed that the migration rate of HSC of miR-9a-5p inhibitor were restrained upon pressure overload.7 Searching and verification for target genes of MiR-9a-5p The luciferase activity of the WT Sirtl 3’-UTR treated by miR-9a-5p mimic was significantly decreased. The protein levels of Sirtl was increased in HSCs with miR-9a-5p inhibitor. Through transfecting with miR-9a-5p mimic, the expression of Sirtl was decreased.8 Sirtl expression analysis in HSCs under pressure and advanced cirrhosis rat liver tissue Through RT-PCR and immunohistochemistry experiments, we verified that Sirtl also was decreased in over-pressured HSCs and liver cirrhosis tissue.9 MiR-9a-5p was involved in PI3K-Akt signaling pathway under pressure overload LY294002, a specific inhibitor of PI3K, could markedly reduce phosphorylation of Akt. Based on the observation that miR-9a-5p was dramatically up-regulated in response to pressure overloading, miR-9a-5p expression was restored by LY294002.Conclusions1 Pressure induced miRNAs were screened in 14 days’activated HSC by Next-generation sequencing and RT-PCR. Among them, MiR-9a-5p significantly expressed by pressure in both HSCs and rat liver fibrosis model. Luciferase report and Western Blot confirmed that Sirtl is the target of MiR-9a-5p, which reduce in the cells and tissues.2 Inhibit the expression of MiR-9a-5p, could significantly inhibit HSC activation, proliferation and migration. Over-expression of Sirtl was able to restrain the activation of HSC. It might be effected through lowed the Sirtl expression quantity.3 Moreover, the results showed that the up-regulation of miR-9a-5p upon pressure overload was associated with the phosphorylation of Akt but not with that of Erk1/2.