| As a class of endogenous RNAs in eukaryotic cells,lnc RNAs are generally larger than 200 nt in length and has not coding protein potential and long open reading frame(ORF)but always has m RNA structural feature.Lnc RNAs can regulate gene expression through a variety of ways.And as one of the major regulation means,lnc RNAs can act as an endogenous sponge to inhibit expression and activities of a number of mi RNAs and then further regulate the downstream signaling pathways.In this study,pc DNA3.1-GAS5 recombinant vector,synthetic si RNAs,mi RNA Mimics and Inhibitors were transfected into 3T3-L1 cells to perform lnc RNA GAS5 and mi R-21a-5p overexpression and knockdown,respectively.CCK-8 and Ed U dyeing were used to determinate the effect of lnc RNA GAS5 on cells proliferation,while Oil Red O staining was use to examine the formation of lipids and q PCR and Western blotting were applied to detect the relative expression of the adipogenic genes m RNA and protein levels.Through prediction software Targetscan and RNAhybrid,we predicted target genes of mi R-21a-5p and luciferase reporter assay was used to validate it,which contributes to explore the specific signaling pathway of lnc RNA GAS5 on 3T3-L1 cells proliferation and differentiation.This study provided basic data for obesity-linked diseases and improvement of animal productivity.The main results of this study are as follows:(1)lnc RNA GAS5 was relatively highly expressed in heart,muscle and fat in4-week-old mice.Contact inhibition and serum starvation could induce the expression of lnc RNA GAS5 during 3T3-L1 cells proliferation.(2)Transfection of pc DNA3.1-GAS5 and mi R-21a-5p Mimics could significantly increased the expression of lnc RNA GAS5 and mi R-21a-5p,respectively(P<0.01).In contrast,transfection of si-GAS5-1/2/3 and mi R-21a-5p Inhibitors could obviously inhibited endogenous lnc RNA GAS5 and mi R-21a-5p expression(P<0.01),and the transfection efficiency could maintain for a long time.(3)The reciprocal repression of lnc RNA GAS5 and mi R-21a-5p was certified by q PCR,and they had the opposite expression pattern during 3T3-L1 preadipocyte differentiation.(4)Overexpression of lnc RNA GAS5 was found to inhibit 3T3-L1 cells proliferation and promote their apoptosis through using CCK-8 kit and Ed U dyeing.However,knockdown of lnc RNA GAS5 expression didn’t have significant effect on3T3-L1 cells proliferation.(5)Overexpression of lnc RNA GAS5 significantly inhibited the accumulation of lipid droplets in cells,reduced the content of triglyceride(P<0.05),the genes PPARγ,FAS,FABP4 and LPL m RNA were significantly decreased(P<0.01,P<0.05),and PPARγ and FABP4 protein levels were also significantly decreased(P<0.05).(6)Target gene prediction showed that mi R-21a-5p could bind with lnc RNA GAS5 and PTEN 3’ UTR,respectively.And dual luciferase reporter assay showed that mi R-21a-5p directly targeted lnc RNA GAS5 and PTEN 3’ UTR and significantly decreased relative luciferase activity(P<0.01).(7)q PCR and Western blotting confirmed that overexpression of lnc RNA GAS5 significantly upregulated PTEN expression in m RNA and protein levels(P<0.01,P<0.05).In contrast,overexpression of mi R-21a-5p dramatically decreased m RNA and protein level of PTEN(P<0.01,P<0.05).In conclusion,this study revealed that lnc RNA GAS5 functions as a sponge could indirectly alleviate impaired PTEN expression by suppressing mi R-21a-5p,thereby inhibiting 3T3-L1 cells proliferation and adipogenic differentiation. |
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