| MiRNA is a non-coding small RNA molecule that negatively regulates target gene expression at post-transcriptional level.It has become an important regulator in metabolism,immunity and cancer.It is received that miRNA can be involved in many important metabolic diseases,such as obesity,atherosclerosis,fatty liver,and diabetes,through regulating the expression of their target genes.Quantitative real-time polymerase chain reaction(qRT-PCR)is the most accurate and convenient method for quantitatively detecting microRNAs.However,appropriate reference genes play an important role in data standardization for the accuracy and stringency of qRT-PCR.Blunt snout bream(Megalobrama amblycephala)is a peculiar herbivorous economic fish in China.Under the condition of artificial cultivation,the disorder of glucose and lipid metabolism for Megalobrama is easy to occur due to the application of high-sugar or high-fat diet.So far,there is no study that has been reported for the selection of miRNA reference genes in blunt snout bream.In view of this,the stability of 10 candidate miRNA reference genes in different stress conditions and tissues was studied in the present study,which might provide scientific basis for the reference genes for miRNAs qRT-PCR in blunt snout bream.Furthermore,miR-30c was applied to investigate lipid metabolism related pathway by injecting miR-30c agomir in vivo.The experiment is mainly divided into two parts:1 Selection of reference genes for miRNA quantitative PCR and its application in miR-34a/Sirtuin-1 mediated energy metabolism in blunt snout bream(Megalobrama amblycephala)The objective of this study was to screen stable reference genes for miRNA RT-qPCR and demonstrate its application in energy metabolism in blunt snout bream(Megalobrama amblycephala)(average weight 15±2 g).The stabilities of ten potential reference genes(miR-21-1-5p,miR-107a-3p,miR-222a-3p,miR-146a-5p,miR-101a-3p,miR-22a-3p,miR-103-3p,miR-456-3p,miR-221-3p and U6(RNU6A))were evaluated using non-reference qRT-PCR in 9 tissues(brain,muscle,liver,skin,spleen,heart,gill,intestine,and eye)under normal condition and in 3 tissues(liver,intestine and spleen)under four stresses(heat stress;ammonia stress;bacterial challenge,and glycolipid stress).This experiment was divided into four stress tests,each of which was sampled at 0 h,3 h,6 h,12 h and 24 h by dynamic sampling method.The sample at 0 h worked as control group.Using GeNorm,NormFinder and RefFinder softwares,we discovered that different tissues and stresses are both important variability factors for the expression stability of miRNAs.After verifying miR-34a/Sirtuin-1 expressions in high-carbohydrate diet-induced blunt snout bream using the relatively stable references,the results showed that the result generated by using miR-221-3p and miR-103-3p as reference genes was consistent with the expected result,while result produced by using the least stable miR-146a-5p as reference genes was not consistent with the result we expected,which means miR-146a could not be used as reference genes in blunt snout bream.This study is the first systematic selection and validation of the optimal miRNA reference gene in blunt snout bream.we eventually identified that the most stable reference gene in this species was miR-221-3p,and the best combination of reference genes were miR-221-3p and miR-103-3p.2 Regulation of miR-30c in Lipid Metabolism in blunt snout bream(Megalobrama amblycephala)The objective of this study is to investigate the regulatory role of miRNA-30c in lipid metabolism in vivo.In this experiment,16 juvenile blunt snout bream(average weight 15±1 g)were selected as experimental animals,which was randomly divided into four groups,each group consisting of four fish.MiR-30c agomir was intraperitoneally injected into each fish.The concentration of miR-30c agomir injection in each group was 0,20,40 and 60 mg/kg,respectively,while the control group was 0 mg/kg.Sampling was carried out after 48 h.The physiological and biochemical indexes of liver and plasma and the expression of lipid metabolism related genes in liver were tested.The results showed that compared with the control group,the contents of cholesterol,triglyceride,low density lipoprotein and free fatty acid decreased significantly after miR-30c agomir injection(p<0.05).And that the expression of lipid metabolism-related genes decreased significantly after the injection of miR-30c agomir(p<0.05)compared with the control group.The results of this study confirmed that miR-30c has a negative regulatory effect on FAS gene,and miR-30c can regulate lipid metabolism by regulating the expression of genes related to lipid metabolism. |
PDF Full Text Request