Screening And Functional Characterization Of Lipid Metabolism-related Micrornas In The Genetically Improved Farmed Tilapia With Fatty Liver Induced By A High-fat Diet

MicroRNAs(miRNAs)play vital roles in modulating diverse metabolic processes in the liver,including lipid metabolism.Overfeeding and dietary nutrient imbalance often induce hepatic steatosis,one of the most common diseases in farmed tilapia.Although fatty liver disease is not an infectious disease.If it did happen,a large-scale farmed tilapia would suffer from this disease.Tilapia with hepatic steatosis grow slowly and they are susceptible to other liver diseases.This disease has resulted in severe economic losses and it has become a serious risk to the tilapia aquaculture industry.Genetically improved farmed tilapia(GIFT,Oreochromis nilolicus)is an important aquaculture species in China.Investigating lipid metabolism-related miRNAs in the liver of GIFT can provide a better understanding of the molecular mechanism underlying the development of hepatic steatosis in fish and provide new insights for reducing the occurrence of fatty liver disease in tilapia farming.In this study,a high fat-diet induced fatty liver GIFT model was constructed by feeding high fat diet to GIFT juveniles.High-throughput sequencing technology was used to detect and quantify miRNA expression levels in GIFT fed either a high-fat diet(HFD)or a normal-fat diet(NFD).Potential lipid metabolism-related miRNAs were selected for further validation.The main results of this study are as follows:1.Construction of HFD induced fatty liver GIFT modelIn this study,two experimental diets containing 8%and 18.5%lipid levels were set as NFD and HFD.GIFT fed with NFD or HFD were sampled respectively for investigating pathological changes during fatty liver formation using biochemical assay,ELISA assay and Microscopic observation at day 20,40 and 60.The results showed that GIFT juveniles fed HFD for 60 days caused a significant decrease(P<0.05)in weight gain(WG)and specific growth rate(SGR),whereas a significant increase(P<0.05)was observed in hepatopancreas somatic index(HSI)and visceral omatic index(VSI).Observion of HE(hematoxylin-eosin)staining and oil red O staining of GIFT microstructure sampled from GIFT fed with experimental diets indicating that high fat feeding caused the appearance of more lipid droplets in the liver of GIFT,which were enlarged.Compared with NFD group,the levels of triglyceride(TG)and total cholesterol(TC)in the liver of GIFT fed with HFD were significantly increased(P<0.05),which was consistent with histopathological observation.During HFD feeding period,the levels of TC,TC,low density lipoprotein-cholesterol(LDL-C),insulin(insulin)and the activity of alanine aminotransferase(ALT)were increased gradually;The activities of hepatic catalase(CAT)and glutathione peroxidase(GSH-Px)were increased firstly and then decreased,superoxide dismutase(SOD)activity did not changed at the beginning,but its activity decreased significantly(P<0.05)on day 60(P<0.05The accumulation of malondialdehyde(MDA)was found in the liver of GIFT fed HFD.In addition,60 days high fat feeding significant changed(P<0.05)hepatic fatty acid composition of GIFT,resulting in an increase in the proportion of polyunsaturated fatty acid(PUFA),saturated fatty acid(SFA)and a decrease in the proportion of monounsaturated fatty aci);d(MUFA).2.Identification and characterization of lipid metabolism-related miRNAs in the liver of GIFTIn this study,a high-throughput sequencing technique was used to construct small RNA libraries from the liver tissues of HFD and NFD groups.After filtering the original sequence data,14085442 and 14006318 high quality clean reads were obtained from HFD and NFD libraries,respectively;Most of the clean reads were from 21-23 nt(nucleotide)in length.After bioinformatics analysis,204 known and 56 novel miRNAs were identified in the two libraries.The ten most abundant miRNAs in the two libraries were identical,they were miR-122,miR-22a-3p,let-7a,miR-21,miR-26a-5p,miR-126a-3p,miR-192,miR-101a,miR-100-5p and let-7g.Differential expression analysis of miRNA revealed that 24 known or novel miRNAs,such as miR-145-5p,miR-205-5p,miR-23a-3p,were significantly up-regulated or down-regulated after 60 days of high fat feeding.The reliability of the sequencing library was validated by quantitative real-time PCR(qRT-PCR).miRanda software was used to predict target genes of nine key miRNAs,including miR-122.Gene Ontology(GO)analysis found that these miRNAs may be involved in the regulation of many basic biological process,including lipid metabolism.3.Expression characteristics of key lipid metabolism genes and miRNAs in the liver of GIFT fed with HFDIn this study,expression of five miRNAs(miR-122,miR-29a,miR-145-5p,miR-34a and miR-205-5p)and their predicted lipid metabolism-related targets were analyzed by qRT-PCR.Target genes were showed as follows:Stearoyl-coenzyme A desaturase(scd)gene,elongation of the very long chain fatty acid protein 6(elovl6)gene,steroid 5 alpha-reductase 2,(srd5a2)gene and acetyl-CoA carboxylase β(acacβ)gene.Molecular analyses revealed that miR-122,miR-29a,miR-145-5p,and miR-205-5p were upregulated,whereas miR-34a was dowhregulated in HFD-fed GIFT.The mRNA levels of hepatic scd,elovl6,and acacβ were decreased and that of hepatic srd5a2 was increased in GIFT fed a HFD.Overall,the results of this study revealed a potential link between miRNAs and fatty liver induced by HFD.4.Study on miR-122 regulation role in lipid metabolismFirstly,this study found that miR-122 was mainly expressed in the the liver of GIFT.The dual luciferase reporter system and eukaryotic expression recombinant plasmid were used to verify that miR-122 can bind to scd mRNA 3’-UTR in HEK293T cells and GIFT liver cells.In the miR-122 overexpression experiment,hepatic scd showed a corresponding down-regulation or up-regulation expression.Under high fat stress,inhibition of endogenous miR-122 expression led to an increase in mRNA levels of hepatic scd,up-regulation of scd expression caused a decrease in the proportion of SFA and an increase in the proportion of MUFA in the liver.In addition,it promoted genes expression involved in lipid synthesis:The transcript levels of sterol-regulatory element binding protein 1(srebp-1)gene and lipoprotein lipase(lpl)gene were increased which resulted in an increase in the synthesis of hepatic TC and TG.In summary,miR-122 can participate in the regulation of lipid metabolism in GIFT by targeting the scd.5 Preliminary study on miR-205-5p regulation role in lipid metabolismIn this study,we first found that miR-205-5p was mainly expressed in the muscle but also expressed in the liver of GIFT by qRT-PCR analysis.Using luciferase reporter assays,we verified the binding site of miR-205-5p in the 3’-UTR of the acacβ mRNA.Furthermore,an in vivo functional analysis of miR-205-5p was performed by injecting GIFT juveniles with the miR-205-5p antagomir.Reduced levels of miR-205-5p in GIFT liver increased acacβ mRNA expression 12h post-injection.miR-205-5p suppression also increased fatty acid synthase and peroxisome proliferator-activated receptor-α mRNA levels 48h and 120h post-injection,respectively.Taken together,our results indicate that miR-205-5p negative regulates hepatic acacβ mRNA expression,and may serve as an important regulator in controlling hepatic lipid metabolism in GIFT.