| Background: MicroRNAs(miRNAs)are a group of non-coding RNA molecules that about 22 nucleotides in length.MiRNAs mostly interact with the 3’ untranslated region(3’UTR)of their target mRNA molecules to regulate protein synthesis and affect multiple signaling pathways.MiRNAs play an important role in tumor development and metabolic diseases,and they possess significant potential and application value in early disease detection,treatment,and monitoring disease progression,as well as organism’s response to various treatments.Recent studies have shown that miR-30b-5p tightly associated with the abnormal lipid metabolism in patients with NAFLD,and decreased expression of miR-30b-5p has been observed in both NAFLD cell model and mouse model.However,the effect of mi R-30b-5p on lipid metabolism is still controversial and the detailed mechanism of miR-30b-5p in the lipid metabolism was remain unclear.The aim of this study was to investigate the role and mechanism of miR-30b-5p on the lipid metabolism in hepatocellular carcinoma Huh-7 cells.Material and Methods: The correlation of intracellular fat content with the expression of miR-30b-5p in Huh-7 cells and HepG2 cells was investigated by treated cells with different concentrations of FFAs(oleic acid: palmitic acid = 2: 1).The total RNAs were extracted from each group of cells and the reverse transcription was conducted.The expression of miR-30b-5p in each group was detected by qRT-PCR.MiR-30b-5p overexpression and inhibition monoclonal stable cell line were established,and the expression of miR-30b-5p in each of the monoclonal cell lines was tested as above description.Each group of monoclonal cell lines were cultured with 0.5mM FFAs for 24 h,the effect of miR-30b-5p on lipid deposition in Huh-7 cells was detected by oil red O staining and TG concentration measurement.Western blot was used to investigate the expression of lipid metabolism-related genes PPAR-α,SREBP-1,and GULT1 in miR-30b-5p overexpressed or inhibited Huh-7 cells.Target genes of miR-30b-5p were predicted using starBase,miRDB databases.The predicted target genes in PITA,microT,miRanda and TargetScan in starBase were extracted and took intersection with the predicted target genes in miRDB.Gene enrichment analysis was made via DAVID and KOBAS,then we choose those lipid metabolism related genes.Western blot and qRT-PCR were used to verify the target genes,and the TargetScan database was used to predict the binding site of the target mRNA to miR-30b-5p.Results: The expression of miR-30b-5p was significant decreased in the FFAs treated Huh-7 cells and HepG2 cells for 24 hours gradually with the concentration of FFAs.Overexpressing mi R-30b-5p in Huh-7 cells decreased intracellular lipid accumulation and the concentration of TG.Inhibiting miR-30b-5p in Huh-7 cells did not change these indexes.Expression of fatty acid oxidation related gene PPAR-α was increased and expression of lipid synthesis related gene SREBP-1 was decreased in the miR-30b-5p overexpressed Huh-7 cells after FFAs treated.And PPAR-α was decreased,SREBP-1 and glucose transport-related genes GLUT1 was increased in the miR-30b-5p inhibited Huh-7 cells after FFAs treated.Target genes of miR-30b-5p were predicted by starBase and miRDB database,and 603 genes were obtained by making intersection of PITA,microT,miRanda,TargetScan and miRDB.After gene enrichment analysis of 603 genes with DAVID and KOBAS,41 genes related to lipid metabolism were obtained.Western blot and qRT-PCR showed that the expression of PPARGC1 A was decreased when miR-30b-5p was overexpressed and the expression of PPARGC1 A was increased when miR-30b-5p was inhibited.Conclusions: In the lipid overload model of Huh-7 cells and HepG2 cells,the expression of miR-30b-5p content was decreased.The stable overexpression of miR-30b-5p in Huh-7 cells can reduce the lipid deposition,up-regulated the expression of PPAR-α and decreased the expression of SREBP-1.In addition,miR-30b-5p could effect the intracellular lipid metabolism by regulate the PPARGC1 A in Huh-7 cells. |
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