Microrna-34c Target ACSL1to Affect Hepatic Stellate Cell Activation

Objective:It is found that miR-34c may play an important role in the process of liver fibrosis inthe previous study in our research group via building DMN rat liver fibrosis model anddetecting the miRNA expression profiling by microarray. Then the target gene predictionsystems speculated that ACSL1may be the possible target of miR-34c. So it is necessaryto further verify whether ACSL1is the target of miR-34c by the luciferase reporter system,detect the expression of miR-34c and ACSL1in hepatic stellate cells (HSCs) of thedifferent states, and thus reveal the role of miR-34c in HSCs activation via regulatingACSL1by downregulating miR-34c gene expression in activated HSCs.Methods:Part I: Rat hepatic stellate cells are extracted and appraised.1.Using enzymatic digestion-density gradient centrifugation method of Friedman:(1)The SD rat was anesthetized for separate the portal vein and intubate, followed byperfusing successively with D-Hank’s solution, pronase and collagenase digestion digestivejuices.(2) After fully digested, the liver was filtrated and the filtrations was centrifugated,the cell suspension was adjusted by Nycodenz and DNase I so that the density is between1.040-1.055g/ml. It is covered with a non-Ga Gass solution for liquid extraction of HSC.(3) After cell counting, HSC in DMEM cell culture medium （20%FBS+1%Penicillin-Streptomycin+0.05%Mycoplasma-OUT）is inoculated in proportion tothe dish.Then the cells placed in CO2incubator and the medium was changed every otherday.2. Observate HSC under microscope at328nm UV excitation.3. detect the expression of Desmin and α-SMA’s in the HSC of2days,14days afterculture with immunofluorescence method.Part II: Detect whether miR-34c target ACSL1and test the expression level ofmiR-34c and ACSL1in different states of the HSC1、Based on the previous work, use Luciferase Reporting System to illustrate thatmiR-34c targets ACSL1; 2、Quantitative Real-time PCR was used to detect the expression of miR-34c and itstarget gene ACSL1in different state of HSCs.Part III:The effect of Downregulations of miR-34c on ACSL1expression and relevantindicators of liver fibrosis in HSCs1. Transfect miR-34c inhibitor into14days’ HSC, simultaneously with twocontrols(the blank group, NC group), and Transfection Indicators Agent was used todetect the transfection efficiency.2. The mRNA expression levels of miR-34c and ACSL1after transfection weredetected by Quantitative Real-time PCR analysis.3. The protein expression levels of ACSL1in HSCs after transfection were detectedby Western-blot method4. Quantitative Real-time PCR and Western-blot were used to measure Type Icollagen and α-SMA mRNA and protein expression levels.Results:1. In this way using Friedman’s method, each SD rat can provide about5-7×107HSCs. In the328nm UV light, HSCs transmit yellow-green fluorescence. The HSCs inquiescency or in activation was detected by Desmin and α-SMA cellularimmunofluorescence and the HSCs purity was more than90%via assay.2. Results of the Luciferase Reporting System confirmed that ACSL1is a target geneof miR-34c; Results of real-time quantitative PCR showed miR-34c expression levels weresignificantly increased (P<0.01). And ACSL1, which was the negative regulated bymiR-34c, was significantly reduced during the HSCs activation (P<0.01).3. Transfected miR-34c inhibitor into activated HCSs, and the transfection efficiencyis up to90%showed by transfection indicator agent. The results of activated HCSs aftermiR-34c inhibitor transfection:(1) results of quantitative Real-time PCR analysis showedthe expression of miR-34c was significantly lower than the blank group and the NC group(P <0.01); and(2) the expression of ACSL1mRNA significantly higher than the controls(P<0.01).(3) Western-blot tests showed that ACSL1protein expression were much morethan the controls.(4) Results of quantitative Real-time PCR and Western-blot tests showedthat both mRNA expressions and protein expression for α-SMAR and Type I collagenwere significantly less than the controls (P <0.01). ConclusionWe extracted HSC from the SD rat liver by using the enzymatic digestion insitu-density gradient centrifugation method of Friedman. The desired cell,identified with highpurity and in good condition,can be provided for subsequent experiments. miR-34cexpression increased during HSCs’ activation, and ACSL1expression, subjected to themiR-34c’s negative regulation, reduced with the activation of HSCs. With reducedexpression of miR-34c, ACSL1expression levels increased in HSCs. Downregulation ofmiR-34c also have an reduction impact on liver fibrosis index α-SMA and collagen Type Iexpression in HSCs. Therefore, miR-34c may regulate the activation state of HSCs andaffect the process of liver fibrosis by targeting ACSL1to regulate some related moleculesof hepatic fibrosis.