The Study Of Dexamethasone Induces Steatosis In Hepatocyte By Regulating LncRNA/miRNA

Part? The expression spectrum analysis of LncRNA/miRNA/m RNA induced by dexamethasone in Hep G2 cellsObjective To establish a model of fatty liver induced by dexamethasone in hepatocytes.Then we want to explore the differences in the expression profiles of non-coding RNA(LncRNA and miRNA)and m RNA induced steatosis by dexamethasone in Hep G2 cell,as well as the possible regulatory network relationship between the three,further select a particular LncRNA-miRNA-m RNA regulation loop,clarify its role in dexamethasone induced steatosis disease.Methods(1)After treating HepG2 cells 24h(n=3)with 300 nmol/L dexamethasone and DMSO(negative control),the oleic acid induction experiment and oil red "O" staining were performed to determine whether the liver cells had steatosis.(2)Using the LncRNA chip(miRCURY ? LNA Arrays v.18.0,Exiqon,Ved-baek,Denmark),miRNA and m RNA chips(the Human lnc RNA Array v3.0)detection for LncRNA,miRNA,m RNA differential expression spectrum,and the differential expression of m RNA of the bioinformatics analysis,including the GO and Pathway analysis.(3)According to the results of bioinformatics analysis,15 LncRNAs,20 miRNAs,and 10 m RNAs were selected for fluorescence quantitative PCR verification.Using the verified LncRNA,miRNA and m RNA and combining the bioinformatics prediction results to construct the ce RNA regulatory network model.(4)From the established ce RNA regulatory network model,the potential ce RNA pathway was selected: LncRNAs-miR-15b-5p-PDK4 signaling pathway.Results(1)After treating HepG2 cells 24 h with 300 nmol/L dexamethasone and DMSO,oil red "O" dyeing experiment results can be observed,compared with control group,the contours of the cells treated by dexamethasone become blunted,it become less closely between cells,have crack,visible in the cell are scattered lipid drops in red,on the edge of the medial to the cell membrane,are too many red lipid droplets in the ring in the intracellular.(2)Microchip test results: compared with untreated Hep G2 cell,there are 528 LncRNAs was up-regulated,and 124 LncRNAs were down-regulated.55 miRNAs were up-regulated,59 miRNAs were down-regulated.527 m RNAs were up-regulated and 128 m RNAs were down-regulated in Hep G2 cell lines treated with dexa-methasone(300 n M).(?2.0 fold-change,P < 0.05).Hierarchical clustering showed differentially expressed in the expression of LncRNA,miRNA,and m RNA were able to distinguish dexamethasone and control two groups of cells.(3)GO analysis showed that the genes with aberrant m RNA expression mainly involved in cellular response to lipids,lipid metabolism and lipid storage regulation,lipase activity,triglyceride metabolism,such as fatty acid biosynthesis of biological processes.Pathway analysis of differentially expressed m RNAs was carried out on,a total of 37 signaling pathways with statistical significance were found,including: fatty acid synthesis,insulin resistance,PPAR signaling pathways,regulating lipid decomposition,carbohydrate metabolism and absorption,steroid hormone biosynthesis and so on.(4)We selected 15 LncRNAs,20 miRNAs,10 m RNAs for q RT-PCR validation,the verification results of PCR are in good agreement with the chip test results.After PCR verification,13 miRNAs,10 m RNAs combined with target gene prediction and expression correlation analysis,the ce RNA action network were constructed,eventually including 12 miRNAs including miR-15b-5p,miR-23a-3p,miR-23b-3p,5 m RNAs: ABHD5,ACSL1,PDK4,CYP7A1,CYP11A1 and 177 LncRNAs including ENST00000608794.The potential ce RNA pathway of interest was selected: ENST00000608794-miR-15b-5p-PDK4 signaling pathway for further study.Conclusion Dexamethasone can interfere with the normal lipid metabolism of hepatocytes,and the expression profiles of LncRNAs,miRNAs and m RNAs can be widely expressed in Hep G2 cells.Part ? The role of LncRNA ENST00000608794-miR-15b-5p-PDK4 signaling pathway in dexamethasone induced fatty liver.Objective For specific LncRNA ENST00000608794-miR-15b-5p-PDK4 conduct the thorough research to clarify its regulating role in dexamethasone induced fatty liver disease,we expected to reveal the mechanism of action of dexamethasone exposure induced steatosis from the perspective of non-coding RNA,thus can provide a new train of thought to alleviate the side effects of this kind of drug.Methods(1)To construct PDK4-3 'UTR and ENST00000608794 luciferin report carrier for double luciferase experiment in order to verify whether miR-15b-5p can be directly combined with PDK4-3' UTR and ENST00000608794.(2)The overexpression virus vector of PDK4 and ENST00000608794 was constructed,and the stable transfection Hep G2 cell line was further established,using fluorescence quantitative PCR and Western-blot to explore whether ENST00000608794 and PDK4 genes could be competitive in inhibiting miR-15b-5p.(3)In the Hep G2 cell line,which PDK4 and ENST00000608794 overexpressed,and in combination with dexamethasone treatment,oleic acid induced experiment and oil red O staining method,we are able to investigate whether ENST00000608794 can inhibit miR-15b-5p,which competitive with PDK4 gene,to regulate PDK4 expression and participate in the process of steatosis induced by dexamethasone.Results(1)Dual luciferase experimental results verify the miR-15b-5p can be directly combined with PDK4.By constructing PDK4 high expression of Hep G2 cell lines,and oleic acid induced and oil red O staining experiments,we found that PDK4 associated with steatosis,in the way of playing a role in the regulation of miR-15b-5p expression level in hepatocyte.(2)The experimental results of luciferase showed that miR-15b-5p could directly bind to the 3 'UTR of ENST00000608794 m RNA,and build the cell lines with high expression of ENST00000608794,as well as the PCR and Western-blot experiments showed that ENST00000608794 could be combined with miR-15b-5p in a competitive combination with PDK4 gene.(3)The expression of LncRNA ENST00000608794 was upregulated after dexamethasone drug treatment.And LncRNA ENST00000608794 expression level can control the degree of liver steatosis,also the realization of this regulation is through competitive binding of miR-15b-5p,thus can protect downstream target genes PDK4 without degradation.Conclusion LncRNA ENST00000608794 by competitive combination genes with PDK4 miR-15b-5p,thus protecting PDK4 lipid metabolism related genes expression from miR-15b-5p negative control,involved in the liver steatosis induced by dexamethasone process control.