Study On The Expression And Function Of MiR-29c In Goose Fatty Liver

Micro RNAs (miRNAs) are class of non-coding single-stranded RNA molecules of about 22 nucleotides. They can reduce translation from protein-coding mRNA transcripts or lead to their degradation by binding to the 3’untranslated regions (3’UTR) of mRNAs.miRNAs as key regulators of gene expression can control biological processes such as growth, metabolism and cancer. Liver as the main place for fatty acid synthesis plays an important role in lipid metabolism.Studies have found that miR-29c can regulate liver fibrosis, and play an important role in the inhibition of liver cancer cell metastasis.However, the expression and regulation of miR-29c and its function in the development of fatty liver is not yet clear. The aim of the present study was to address this.The main results are as follows:1. The expression of miR-29c in the liver, muscle and fat was inhibited by overfeeding. In this study, real-time PCR was used to detect the expression of miR-29c in the liver, muscle and fat of both overfed and normally-fed geese. The data showed that the expression of miR-29c in these tissues was significantly inhibited by overfeeding, suggesting that it plays an important role in the development of goose fatty liver.2. Prediction and screening of miR-29c target genes. The target genes of miR-29c was predicted by bioinformatics tools (TargetScan, miRDB and Microcosm) with chicken genome, followed by combination with the results from previous studies of goose fatty liver. The target genes including COL3A1, SGK1, INSIG1, DDX3X, PPM1D and TNFAIP3 were selected for further investigation. Real-time PCR analysis indicated that the expression of COL3A1, SGK1, and INSIG1 was significantly induced in the livers of the overfed vs. normally fed geese, while DDX3X, PPM1D and TNFAIP3 was not. As the expression of miRNAs is generally opposite to that of its target gene, we inferred that COL3A1, SGK1 and INSIG1 were most likely the target genes of miR-29c.3. Verification of miR-29c target genes. First, we designed the primers of 3’UTR for the candidate target genes of miR-29c, followed by PCR amplification, cloning and sequencing of these 3’UTR sequences.The pMIR-REPORT system was used to construct the expression vectors of the 3’UTR sequences. We also designed miR-29c mimics based on the mature sequence of miR-29c for verification of miR-29c target genes. The mimics were transfected into CHO cells, and their activities were evaluated by dual luciferase reporter system. The result demonstrated COL3A1, SGK1, and INSIG1 were indeed the target genes of miR-29c. Thus, miR-29c may affect the formation of goose fatty liver through these target genes.4. The effect on the expressin of the target genes by miR-29c in goose primary hepatocytes. The miR-29c mimics, inhibitor and negative control were transfected into goose primary hepatocytes, and the expression of the target genes were detected by quantitative PCR. Data indicated that the expression of COL3A1 and INSIG1 was regulated by miR-29c in goose liver cells, while the expression of SGK1 was propably interfered by other factors. Moreover, the expression of COL3A1 and INSIGl was more easily affected by miR-29c especially when the expression of miR-29c was suppressed.5. The effect of fatty liver-related factors on the expression of miR-29c in goose primary hepatocytes.In this study, goose primary hepatocytes were treated with different dosages of glucose, insulin, and fatty acids and the expression of miR-29c was subsequently determined. The results showed that 100mmol/L glucose and 0.5mmol/L palmitate reduced the expression of miR-29c significantly compared to control cells, but 100nmol/L insulin and 0.5mmol/L sodium oleate had no effect on the expression of miR-29c. These findings suggest that the expression of miR-29c is influenced by high glucose and fatty acid, and support miR-29c is involved in the development of goose fatty liver.6. The regulation of INSIG1 and COL3A1 by glucose was mediated by miR-29c in goose primary hepatoctyes. miR-29c inhibitor or negative control were separately transfected into goose primary hepatocyte, followed by treating the cells with glucose. The expression of INSIG1 and COL3A1 was determined by quantitative PCR. The results showed that the induction of INSIG1 by 100 mmol/L glucose was further enhanced by miR-29c inhibitor, while the inhibition of COL3A1 by glucose was rescued by miR-29c inhibitor. These findings suggested that the regulation of INSIG1 and COL3A1 by glucose was mediated by miR-29c in goose primary hepatocytes.7、Prediction and initial verification of transcription factor of miR-29c. First, upstream sequences of miR-29c was amplified by PCR with primers designed, followed by cloning and sequencing the amplifications. Using the upstream sequence, we predicted transcription factors) including RFX1 for miR-29c using Gene Regulation software. Retinoic acid as regulator of RFX1 activity was used to treat goose primary hepatocytes. Data indicated the expression of miR-29c was upregulated by 10μmol/Lretinoic acid, suggesting RFX1 is possible a transcription factor of miR-29c.