| microRNA (miRNA) are small non-coding RNAs (usually with the length of 19-25 nt) that are found in a variety of eukaryotes and viruses, and usually they have functions in the transcriptional and post-transcriptional regulation of gene expression, by promoting mRNA degradation (usually in plants) or inhibiting protein translation (usually in mammals) via base-pairing with complementary or incomplete complementary sequences within mRNA molecules. miRNAs are well conserved among different species and can be involved in the regulation of a series of biological processes. Recently, numerous studies on human and other animal models have found that many miRNAs play positive or negative regulatory roles in fat metabolism and adipocyte differentiation and they have been gradually shown the future prospects of anti-obesity clinical application.Unfortunately, so far most of the studies about miRNAs are limited at the basic theoretical levels and few research was focused on its application in biomedicine and agricultural production, which dues to the lack of suitable nucleic acid drug delivery systems. Therefore, the safe, stable, efficient and specific delivery vehicle is the key technology for successful clinical practice of nucleic acid drugs in clinical trials and agricultural production. Nowadays, extracellular vesicles (EVs) are used as new transport tools of nucleic acid drugs and they have been the focus of biomedical research community.So far, studies on the regulation of miRNAs in fat metabolism are mainly in human and other rodent models, few study is focused on porcine. Therefore, in the present study, we want to screen the miRNAs that are important for porcine fat metabolism through two different fat deposition models and varify their functions, in addition, we want to package one of the screened miRNAs (miRNA-130b) into micro vesicle (MV) (one kind of EVs) and to further validate the function of MV packaged miRNA-130b through in vitro and in vivo experiments, so as to provide theoretical basis for the biomedicine and agricultural production.1 Screening of the key miRNA molecules involved in porcine fat metabolism using an in vivo experimental model.Previous studies have shown that maternal low protein diet can affect offspring lipid deposition. Therefore, in the present study,14 primiparous purebred Meishan sows with average weight of 36.1 ± 1.8 kg were randomly separated into low-protein (LP) and standard-protein (SP) groups, SP sows were fed with 12% and 14% crude protein diet while the LP sows were fed with 6% and 7% during gestation and lactation respectively. Litter size was adjusted to 7-8 pigs per litter at 24 h post farrowing in the same group. Newborn piglets were weaned at 35 d of age.Male pigs were killed at weaning stage and determined the body weight and backfat levels, as well as the serum TG and FFA concentrations. RT-PCR and Western blotting methods were used to determine the expression of PPAR-y and C/EBP-β. Then we used bioinformatics to predict the possible miRNAs targeting PPAR-y and C/EBP-β and analyzed the homology of the target sites (Seeding region), furthermore, we determined the miRNA expression abundance by RT-PCR. The results suggest that although the serum TG concentration did not change between the two groups, the piglets body weight, back fat thickness and index of back fat thickness were significantly decreased (P< 0.05) in the LP group compared with the SP group, furthermore, the serum FFA concentration also has a downward trend (P= 0.083). Although mRNA expression of PPAR-y and C/EBP-β in the subcutaneous fat showed no significant change between the two groups, the protein level of PPAR-y was significantly decreased (P< 0.05) in the LP group, and C/EBP-β protein expression was also decreased, though not significant (P= 0.056). Furthermore, miRNA expression analysis showed that miRNA-130b, targeting the PPAR-y 3’-UTR, and miR-374b, targeting the C/EBP-β 3’-UTR, were both significantly increased (P< 0.05) in the LP group compared with the SP group, while other candidate regulatory miRNAs were expressed similarly in both groups. And the binding sequences of miRNA-130b and miRNA-374b on its target gene 3’-UTR has a higher conservation among different species.The above results showed that we employed the fat deposition reduced weaned offspring which was induced by the maternal low protein diets as animal model and successfully screened the miRNA-130b and miRNA-374b which play an important regulatory role in adipocyte differentiation, in addition, we found that miRNA-130b and miRNA-374b were able to bind and inhibit PPAR-y and C/EBP-β protein expression and thus play a role in reducing fat deposition.2 Screening of the key miRNA molecules involved in lipid droplet deposition using an in vitro experimental model.In the previous study, we have known that glucocorticoids (GCs) can reduce lipid deposition. Therefore, in the present study, we use GCs to treat the primary cultured porcine adipocytes and screen the key miRNAs involved in adipocyte differentiation and fat deposition basing on the in vitro model. SV cells were separated from subcutaneous adipose tissue of weaned Meishan piglets. Cells were cultured to 80% confluence followed by treatments with 10-6 M dexamethasone, and control group was treated with nothing. MTT was used to determine the effect of 10-6 M dexamethasone on cell viability and TG content was measured by Oil-red O staining, RT-PCR method was used to determine the mRNA expression of genes related to adipogeensis while western blotting was emplyed to evaluate PPAR-γ and C/EBP-β protein levels, furthermore, the expression of miRNAs targeting PPAR-γ and C/EBP-p mRNA 3’-UTR was also measured by RT-PCR. The present results suggest that TG content in cultured adipocytes was significantly increased (P< 0.05) after 10-6 M dexamthasone treatment for 48 h compared with control group while the cell viability did not change among the four groups (Control; 10-6 M Dexamethasone; 10-6 M RU486; 10-6 M Dexamethasone+10-6 M RU486). RT-PCR results showed that 10-6 dexamethasone could significantly up-regulate mRNA expression of perilipin and PPAR-y (P< 0.05), however, C/EBP-β mRNA expression was significantly down-regulated (P< 0.05). We further determined the expression of miRNAs predicted to directly target PPAR-γ and C/EBP-β and found that miRNA-374a and miRNA-374b, targeting the C/EBP-β 3’-UTR, were significantly increased (P< 0.05). Furthermore, miR-128 and miR-130b, targeting the PPAR-γ 3’-UTR, were also significantly increased (P <0.05).These results suggest that we used the primary cutured porcine adipocytes which was treated by GCs as in vitro model and successfully screened the key miRNA-128, miRNA-130b and miRNA-374a/b which can play an important role in adipocyte differentiation and fat deposition.3 Validation of the role of miRNA-130b and miRNA-374b by using dual-luciferase reporter assay.In the present study, we employed dual-luciferase reporter assay to verify the role of miRNAs in regulating of its target gene expression.3’-UTR of PPAR-y and C/EBP-β mRNA was amplified by PCR from porcine genomic DNA using gene specific primers which has a XbaI restriction enzyme site. Each amplified 3’-UTR was cloned into the firefly luciferase gene in the pGL3 luciferase reporter vectors at the XbaI restriction sites. Pre-miRNA or a negative control (Scrambled control, miRNA-SC) was cloned into pSilencer 3.0-H1 siRNA expression vectors between BamHI and HindⅢ restriction sites. And then,3’-UTR-pGL3 firefly luciferase reporter vector and miRNA over-expression vectors or miRNA-SC were co-transfected with pRL-TK renilla luciferase vector into human cervical cancer cell line Hela-229 cells.24 h and 48 h post-transfection, firefly and Renilla luciferase activities were determined using the dual-luciferase reporter assay system. Results of dual-luciferase reporter assay showed that over-expression of ssc-miR-130b significantly repressed (P< 0.05) the activity of firefly luciferase reporter gene followed by 3’-UTR of PPAR-y and over-expression of ssc-miR-374b repressed the activity of firefly luciferase followed by 3’-UTR of C/EBP-β.Dual luciferase activity assay results indicated that miR-130b directly recognised and bound to the 3’-UTR of PPAR-y and thereby suppressed PPAR-y gene expression. furthermore, miR-374b directly recognised and bound to the 3’-UTR of C/EBP-β and thereby suppressed C/EBP-β gene expression.4 Preparation and function validation of MV packaged miRNA-130b in in vitro model.In our previous study we found that miRNA-130b suppressed fat deposition by inhibiting PPAR-y expression. In order to demonstrate whether miRNA-130b can be packaged into microvesicles and function as an endogenous form of miRNA-130b in recipient cells, we transfected HeLa-229 cells with miRNA-130b over-expression plasmid to over-express miRNA-130b, and we found that miRNA-130b expression in HeLa-229 cells, media, MV and MV-free media were all increased significantly (P< 0.05), which indicated that HeLa-229 cells were able to selectively package miRNA-130b into MV under miRNA-130b over-expression and actively secrete the miRNA-130b enriched MV into the culture media. We further verified that MV enriched with miRNA-130b contained elevated concentrations of Argonaute 2 (P< 0.05) and heat shock protein 90a (P< 0.05) which are known to protect the circulating miRNAs from degradation. Exposure of primary cultured porcine adipocytes to purified, miRNA-130b-enriched MV resulted in a significant up-regulation of TG concentration (P< 0.05) while the cell viability did not change. After the administration of miRNA-130b-MV for 48 h, miRNA-130b expression was significantly up-regulated while the PPAR-y expression was inhibited (P<0.05); in addition, the expression of adipogenesis and lipogenesis related genes also changed, FAS (Fatty acid synthase), Perilipin, FTO (Fat mass and obesity-associated gene) and GR mRNA expression were significantly down-regulated (P< 0.05) while HSL (Hormone sensitive lipase) mRNA expression was increased significantly (P< 0.05).Taken together, our results suggested that Hela-229 cells were able to be successfully packaged miRNA-130b into MV in the case of miRNA-130b over-expression. In addition, we found that MV-shuttled miRNA-130b could be delivered into the primary cultured adipocytes and reduced fat deposition in recipient adipocytes by targeting PPAR-y. We also showed that MV might provide an effective transport systems for the deliver of miRNAs for therapeutic use.5 Functional validation of MV packaged miRNA-130b in in vivo model.In order to further investigate whether the MV-shuttled miRNA-130b could exert its biological function in in vivo model and reduces fat deposition, we employed SPF C57BL/6N male mice (three weeks) as experimental animal models and the mice were randomly divided into two groups:the control group were fed a normal fat diet (MD 10%, n= 12); obesity induced group were fed high fat diet (MD 45%, n= 36). After fed for 8 weeks, body weight and glucose tolerance were detected. After we confirmed that the obese mice model was successfully established, the obesity mice were randomly divided into two groups, one was high-fat group with tail vein injection of miR-130b-MV (HF-130b-MV); the control group was high-fat group with intravenously injected miR-SC-MV (HF-SC-MV), once every two days,200μL each time (approximately 200 p.g MV total protein), then got samples after 10 days injection. The results suggested that the body weight of high fat diet group were significantly increased (P< 0.001) after feeding for eight weeks when compared with control group, furthermore, glucose resistance was also increased (P< 0.05). However, when compared with HF-SC-MV group, miR-130b-MV injection for 10 days could reduce insulin resistance caused by HF diet. In addition, miR-130b-MVs injection could significantly reduce the body weight (P< 0.05) as well as epididymal fat (P< 0.05) and gastrocnemius weight (P< 0.05); however, no change was observed in liver weight; furthermore, when compared with HF-SC-MV group, miR-130b-MV injection reduced the epididymal fat weight/body weight, though not significant (P= 0.063); liver weight/body weight was significantly increased (P< 0.05) while gastrocnemius weight/body weight did not change.These results revealed that MV-shuttled miRNA-130b could also has function in in vivo model and reduced fat deposition, however, the specific molecular mechanisms need to be further explored. |
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