The Construction Of Lentiviruses Vector Of MiR-196b-5p And The Impact On Adipocyte And Osteoblast Differentiation

Objective: Micro RNAs(miRNAs)are a kind of short(about 22 nt)endogenous non-coding RNA that via fully or partially base pairing with m RNA degradate or inhibit its translation,regulate the expression of target genes in the post-transcription level.Mi RNAs participate in many types of biological processes,including cell division,cell differentiation,cell apoptosis,metabolism,cancer and so on.Many studies have shown that miRNAs play an important role in mesenchymal stem cells(MSCs)differentiation,and exists a reciprocal and inverse relationship between adipogenesis and osteogenesis.In this study,we aimed to investigate the biological function of mi R-196b-5p in regulating adipocyte and osteoblast differentiation,and preliminarily elucidated the mechanism about adipogenesis.Methods: 1 ? By using quantitative q RT-PCR,we tested the expression of mi R-196b-5p in the mouse bone marrow mesenchymal stem cells(BMSCs)and bone marrow stromal cell line ST2 cells after treated with adipogenic or osteogenic medium.2?We made the lentiviruses plasmid that either overexpression mi R-196b-5p precursor(mi R-196b-5p LV)or knockdown endogenous mi R-196b-5p(mi R-196b-5p sponge).3?Mouse BMSCs and ST2 cells were infected with the lentiviruses.The effects of mi R-196b-5p LV and mi R-196b-5p sponge on adipogenesis were detected by q RT-PCR?Western Blotting and Oil-red O staining,qRT-PCR ?Western blotting and ALP staining were conducted to detect osteogenesis.4?The potential target genes of mi R-196b-5p were predicted using online tools.Then the 3 'UTR fragment of the target gene(Tgfbr1-3?UTR)was PCR-amplified and cloned into luciferase reporter vector.The luciferase activity of Tgfbr1-3?UTR was detected by dual-luciferase reporter gene assay.mi R-196b-5p mimics and mi R-196b-5p inhibitor were transfected ST2 cells respectively,and the expression quantity of TGFBR1 were measured.mi R-196b-5p mimics and Tgfbr1 were cotransfected to conduct cell rescue experiment.5?4-week-old male C57BL/6J mice were fed with high fat diet and obese mice model was constructed.The body weights of mice in each group were recorded and the mice were tail vein injected mi R-196b-5p sponge or negative control lentiviruses.Weighing the inguinal fat and epididymal fat weight after the model was successfully built,and detecting the m RNA expression changes of adipogenesis related genes by qRT-PCR about epididymal fat.Moreover,biochemical kits were used to examine glucose,insulin,triglyceride and free fatty acid levels in serum.These experiments help to determine the impact of knockdown endogenous mi R-196b-5p on obesity.Results: 1?qRT-PCR showed that the expression of mi R-196b-5p in mouse BMSCs and bone marrow stromal cell line ST2 cells were increased after adipogenic treatment,conversely,the expression of mi R-196b-5p in mouse BMSCs and ST2 cells were decreased after osteogenic treatment.2?We constructed the lentiviruses that either overexpression mi R-196b-5p precursor or knockdown endogenous mi R-196b-5p successfully.Mouse BMSCs and ST2 cells were infected with mi R-196b-5p LV and followed adipogenic treatment increased Oil-red O-positive adipocyte formation,enhanced the expression of PPAR?,C/EBP?,a P2,and adipsin;conversely,infected with mi R-196b-5p sponge and followed adipogenic treatment reduced Oil-red O-positive adipocyte formation,attenuated the expression of PPAR?,C/EBP?,a P2,and adipsin.Mouse BMSCs and ST2 cells were infected with mi R-196b-5p LV and followed osteogenic treatment made ALP staining shallowed,the expression of Osterix,ALp,Osteocalcin and Bsp were down-regulated;conversely,infected with mi R-196b-5p sponge and followed osteogenic treatment made ALP staining deepen,the expression of Osterix,ALp,Osteocalcin and Bsp were up-regulated.3?Dual-luciferase reporter gene assay revealed that Tgfbr1 is likely to be target of mi R-196b-5p.mi R-196b-5p mimics could increase the expression quantity of TGFBR1 when mi R-196b-5p inhibitor decreased it.Tgfbr1 could suppress adipogenic differentiation in ST2 cells,and mi R-196b-5p mimics could save it.4?Obesity mouse model was successfully built after 8 weeks by feeding with high fat diet.By contrast,tail vein injected mi R-196b-5p sponge lentiviruses reduced the weight,the groin fat and epididymal fat weights also lower than the high fat control.Meanwhile,the m RNA expression changes of adipogenesis related genes about epididymal fat also lower than the high fat control.Furthermore,high fat diet significantly increased the serum level of glucose,insulin,triglycerides and free fatty acid,while mi R-196b-5p sponge lentiviruses decreased them.Conclusion: 1 ? The expression of mi R-196b-5p in the mouse bone marrow mesenchymal stem cells(BMSCs)and bone marrow stromal cell line ST2 cells were increased after adipogenic treatment,and decreased after osteogenic treatment.2?mi R-196b-5p LV promoted the differentiation of the mouse BMSCs and ST2 cells into adipocytes,and suppressed they differentiated into osteoblasts;conversely,mi R-196b-5p sponge suppressed they differentiated into adipocytes,and promoted they differentiated into osteoblasts.3 ? mi R-196b-5p may promote adipocyte differentiation through targeting Tgfbr1.4?mi R-196b-5p sponge lentiviruses could relief of serum biochemical indices,which caused by high fat diet,and improved the insulin sensitivity,inhibited adipocyte size and the generation of obesity.